Oxidation reduction sensitive green fluorescent protein variants

ABSTRACT

The disclosure provides proteins that can be used to determine the redox status of an environment (such as the environment within a cell or subcellular compartment). These proteins are green fluorescent protein (GFP) variants (also referred to as redox sensitive GFP (rosGFP) mutants), which have been engineering to have two cysteine amino acids near the chromophore and within disulfide bonding distance of each other. Also provided are nucleic acid molecules that encode rosGFPs, vectors containing such encoding molecules, and cells transformed therewith. The disclosure further provides methods of using the rosGFPs (and encoding molecules) to analyze the redox status of an environment, such as a cell, or a subcellular compartment within a cell. In certain embodiments, both redox status and pH are analyzed concurrently.

STATEMENT REGARDING GOVERNMENT FUNDING

[0001] Aspects of the research leading to this disclosure were supportedby the following government agencies under the identified grants:National Institutes of Health Training Grant for GTH I/D Serial No.:GM07759-22; and National Institutes of Health Grant I/D Serial No.:GM42618-10. The government may have certain rights in this invention.

FIELD

[0002] The present disclosure relates to the field of geneticengineering, and in particular to green fluorescent protein (GFP)mutants that can be used to detect oxidation-reduction state, or achange in oxidation-reduction state.

BACKGROUND

[0003] The green fluorescent protein (GFP) from the Pacific Northwestjellyfish, Aequorea victoria, has been used extensively in molecular andcell biology as a fluorescent marker. It is a 238 amino acid proteinthat generates its own fluorescent chromophore. The spontaneousgeneration of the chromophore is achieved by cyclization of the internalSer65-Tyr66-Gly67 sequence followed by oxidation of Tyr 66 in thepresence of molecular oxygen (Heim et at, Proc. Natl. Acad. Sci. USA91:12501-12504, 1994). The overall fold of the protein consists of an11-stranded β-barrel capped by α-helices at both ends and contains acoaxial α-helix from which the chromophore is generated (Brejc et al.,Proc. Natl. Acad Sci. USA 94:2306-2311, 1997; Orm{umlaut over (o )}etal., Science 273:1392-1395, 1996; Yang et al., Nat. Biotech:14:1246-1251, 1996). GFP is unique among light emitting proteins,because it does not require the presence of any cofactors or substratesfor the production of green light.

[0004] Wild-type GFP has absorption maxima at 398 and 475 nm (Morise etal., Biochemistry 13:2656-2662, 1974). Excitation at either of thesewavelengths leads to emission of green light at 508 nm (Morise et al.,1974). The usefulness of GFP has been greatly enhanced by theavailability of mutants with a broad range of absorption and emissionmaxima (Heim et al., Proc. Natl. Acad. Sci. USA 91:12501-12504, 1994;Orm{umlaut over (o )}et al., Science 273:1392-1395, 1996). These mutantshave made possible multicolor reporting of cellular processes byallowing for the simultaneous observation of two or more gene productslabeled with different colored GFP variants (Rizzuto et al, Curr. Biol6:183-188, 1996). In addition, fluorescence resonance energy transfer(FRET) experiments using different colored GFP's have been used to studyprotein-protein interactions in vivo (Heim et al, Curr. Biol 6:178-182,1996; Mitra et al., Gene 173:13-17, 1996).

[0005] More recently, GFP variants have been shown to be sensitive to pH(Wachter et al., Biochemistry 36:9759-9765, 1997; Elsliger et al.,Biochemistry 38:5296-5301, 1999). As a consequence, they have been usedas noninvasive intracellular pH indicators. For instance, Kneen et al.employed the GFP mutant S65T/F64L to determine the pH of the cytoplasmof CHO and LLC-PK1 cell lines (Kneen et al., Biophys. J. 74:1591-1599,1998). Since GFP is genetically encoded, it can be specifically targetedto various subcellular compartments, which is a task not possible withsmall molecule fluorescent dyes (Llopis et al., Proc. Natl. Acad. Sci.USA 95:6803-6808, 1998). Therefore, Llopis and co-workers used the GFPvariant S65G/S72A/T302Y/H231L, which has an increased pK_(a), to measurethe alkaline pH of mitochondria golgi, and the cytosol of HeLa cells andrat neonatal cardiomyocytes (Llopis et al., 1998). These reports werethe first to show that GFP variants could be used as biosensors and notjust simple fluorescent markers. However, more recently GFP has beenshown to be sensitive to halide ions and through a fusion withcalmodulin, GFP's fluorescence can also vary in response to calcium ionconcentration (Wachter at., Curr.Biol. 9:R628-R629, 1999; Miyawaki etal., Proc. Natl. Acad Si. USA 96:2135-2140, 1999).

[0006] Oxidation-reduction (redox) processes are very important inliving organisms. The formation of disulfide bonds during proteinfolding relies upon a well maintained redox buffering system ofglutathione and oxidized glutathione (Carothers et al., Arch. Biochem.Biophys. 268:409-425, 1989). There also exists a thioredoxin-like familyof enzymes that catalyze the formation and isomerization of disulfidebonds in proteins (Debarbieux and Beckwith, Cell 99:117-119, 1999). Inaddition, redox signaling during apoptosis has been implicated inactivating mitochondrial permeability transition, leading to cytochromec release (Hall, Eur. J. Clin. Invest. 29:238-245, 1999). Redox changesin the form of cellular oxidation have also been suggested to be a finalstep in the apoptotic process leading to degradation of apoptotic bodies(Cai and Jones, J. Bioenerg. Blomemb. 31:327-334, 1999). Given theimportance of in vivo processes such as protein folding and apoptosisthat are dependant upon redox status, a non-invasive, convenient methodfor studying redox changes within living cells is needed.

[0007] Current methods of determining in vivo redox status have manylimitations. Many present techniques require cells to be harvestedbefore their contents can be analyzed. This type of procedure is notonly very invasive but is also not a very accurate measure of the invivo state of the cells. Moreover, it would be impossible with thistechnique to monitor redox changes within the same cell over a period oftime. Recently, Keese et al. (Keese et al, FEBS Lett. 447:135-138, 1999)have developed an indicator of redox state in which glutathionereductase crystals were microinjected into the cytosol of humanfibroblasts, and by detecting a color change of the crystals, they wereable to determine the redox potential of the cytosol to be more reducingthan −0.270 V. While this method may allow redox determination withinsingle living cells, the cumbersome nature of the technique is still amajor drawback. The most reasonable protocol for determining redoxstatus is probably still that of Hwang et al. (Hwang et al., Science257:1496-1502, 1992). They employed the tetrapeptideN-Acetyl-Asn-Tyr-Thr-Cys-NH₂ to measure the ratio of thiol to disulfidein the cytosol and secretory pathway of cultured cells. They concludedthat the cytosol is more reducing than the secretory pathway with anapproximate redox potential of −0.221 to −0236 V for the cytosolcompared to −0.170 to −0.185 V for the secretory pathway. However, ismethod still required harvesting of the cells and like all the othermethods, it is very labor intensive. Moreover, this technique determinedredox potentials based only upon the ratio of reduced glutathione (GSH)to oxidized glutathione (GSSG), potentially ignoring other redoxbuffering components.

SUMMARY OF THE DISCLOSURE

[0008] To overcome disadvantages of available methods for determiningredox status in cells, GFP mutants (also referred to as redox sensitiveGFP (rosGFP) variants) have been designed and are described herein,which can detect or “sense” changes in oxidation-reduction potentials.The rosGFP variants have been engineering to have two cysteine aminoacids near the chromophore and within disulfide bonding distance of eachother.

[0009] Examples of the provided GFP variants have ratiometricdual-excitation fluorescent properties as a function of redox state,with apparent redox potentials of −0.272 to −0.299 V.

[0010] Specific embodiments include rosGFP mutants that differ fromwild-type GFP in that they comprise at least the following amino acidsubstitutions:

[0011] (a) S147C/Q204C

[0012] (b) S65T/S147C/Q204C

[0013] (c) N149C/S202C

[0014] (d) S65T/N149C/S202C

[0015] (e) S147C/N149C/S202C/Q204C

[0016] (f) S65T/S147C/N149C/S202C/Q204C

[0017] The rosGFP mutants that include the S65T substitution aresensitive to pH as well as redox status. Particular provided mutationproteins include those referred to herein as rosGFP1, rosGFP2, rosGFP3,rosGFP4, rosGFP5, and rosGFP6.

[0018] Also provided are nucleic acid molecules encoding rosGFPs,including the specific listed rosGFPs. Optionally, these nucleic acidmolecules can be functionally linked to expression control sequence(s)(such as a promoter), and/or integrated into a vector. Nucleic acidmolecules encoding a rosGFP can be used to transform host cells (such asbacterial, plant, or animal cells); such transformed cells are alsoprovided.

[0019] The disclosure also provides methods of using rosGFPs to analyzethe redox status of a cell, or a subcellular compartment within a cell.In certain embodiments, both the redox status and pH of the cell (orsubcellular compartment or other environment) are monitoredconcurrently.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 is a fluorescence spectra graph, which shows how thefluorescence of rosGFP2 varies in response to changes in redoxpotential. The spectra show two excitation peaks, one near 400 nm andthe other at about 490 nm, with a clear isosbestic point separatingthem.

[0021]FIG. 2 shows the titration of rosGFP2 with dithiothreitol. Theapparent redox potential is −0.279 volts

[0022]FIG. 3 is a graph showing the in vivo redox changes influorescence intensity of rosGFP2, in response to the addition ofvitamin in K₃. After 30 total minutes, the addition of dithiothreitolelicits the opposite response as indicated by the reduced ratio.

[0023]FIG. 4 shows an SDS-PAGE analysis that reveals the intracellulardisulfide linkage in rosGFP2. Lanes 1-6, control (C48S/S65T) and lanes 8-13 rosGFP2 were incubated with 1 μM CuCl2 (with or without 2 mMN-ethylmaleimide; lanes 2, 3, 11, 12) or with 1 mM DTT (with or without2 mM N-ethylmaleimide; lanes 5, 6, 8, 9). Lane 7 shows approximatemolecular weights in kDa. Lanes 4 and 10 were empty.

[0024]FIG. 5. Absorbance and fluorescence excitation spectra of rosGFP2at various redox states. The absorbance spectra (A) show the conversionof the neutral (band A; 400 mn) to the anionic (band B; 490 nm)chromophore species over time in the presence of 1 mM DTT. Band A ismaximized under oxidizing conditions, whereas band B is favored underreducing conditions. Fluorescence spectra (B) were collected at variousredox potentials and also show the interconversion of chromophore chargestates. Absorbance and fluorescence spectra were both normalized to theintensity of the fully reduced protein.

[0025]FIG. 6. Fluorescence excitation spectra of rosGFP4 as a functionof redox potential. The entire spectrum (A) shows the redox potentialdependence on the excitation spectra of rosGFP4. Expanded the regionaround 400 nm (B), reveals a well resolved isosbestic point.Fluorescence intensity values were normalized to the maximum intensityat E_(o)′ −0.320 V and emission was monitored at 510 nm.

[0026]FIG. 7. Fluorescence excitation spectra of rosGFP6 as a functionof redox potential. Fluorescence intensity values were normalized to themaximum intensity at E_(o)′ −0.310 V and emission was monitored awayfrom the peak at 535 nm.

[0027]FIG. 8. Absorbance and fluorescence excitation spectra of oxidizedrosGFP2 as a function of pH. Absorbance scans (A) were taken on samplesof rosGFP2 containing 0.5 μM CuCl₂ at the indicated pHs. These sampleswere then diluted in the same buffer and their fluorescence excitationspectra (B) were collected. Fluorescence intensity values werenormalized to the maximum intensity at pH 9.0 and emission was monitoredat 510 nm.

[0028]FIG. 9. pH titration of oxidized and reduced rosGFP2. Absorbancevalues at 490 nm (band B) were plotted versus pH for oxidized (A) andreduced (B) rosGFP2. The data were then fitted to a titration curve witha single pK_(a) value,

[0029]FIG. 10. Absorbance and fluorescence excitation spectra of reducedrosGFP2 as a function of pH. Absorbance scans (A) were taken on samplesof rosGFP2 containing 1 mM DTT at the indicated pHs. These samples werethen diluted in the same buffer and their fluorescence excitationspectra (B) were collected. Fluorescence intensity values werenormalized to the maximum intensity at pH 9.0 and emission was monitoredat 510 nm. Absorbance readings around 280 nm are greatly altered due tothe presence of DTT and thus are not shown.

[0030]FIG. 11. Fluorescence excitation spectra of rosGFP1 at variousredox potentials. Fluorescence intensity values were normalized to themaximum intensity at E_(o)″ −0.320 V and emission was monitored at 510nm.

[0031]FIG. 12. Fluorescence excitation spectra of rosGFP3 at variousredox potentials. The entire spectrum (A) shows the redox potentialdependence on the excitation spectra of rosGFP3. Expanded the regionaround 405 nm (B), reveals the existence of an isosbestic point.Fluorescence intensity values were normalized to the maximum intensityat E_(o)″ −0.330 V and emission was monitored at 510 nm.

[0032]FIG. 13. Fluorescence excitation spectra of rosGFP5 at variousredox potentials. Fluorescence intensity values were normalized to themaximum intensity at E_(o)″ −0.330 V and emission was monitored off thepeak at 535 nm.

[0033]FIG. 14. A fluorescence excitation ratio results in thecancellation of pH artifacts. In the oxidized (A) or reduced (B) state,a ratio of fluorescence intensities at various excitation wavelengths ofrosGFP2 is independent of pH.

[0034]FIG. 15. Dual-emission characteristics of rosGFP2. Excitation at400 nm results in emission peaks centered near 450 and 510 nm, whichhave an opposite response to pH changes.

[0035]FIG. 16. A fluorescence emission ratio results in the cancellationof redox potential changes on pH determination. The fluorescenceemission spectra (A) of rosGFP2 were collected at various redoxpotentials (ratios of DTT and DTT_(ox)) and at a constant pH of 6.0.Plotting the ratio of the two emission peaks results in a constant ratioover a large range of redox states (B). The dashed lines in B representthe maximum and minimum ratios to illustrate the possible dynamic rangeof rosGFP2 as a function of pH.

[0036]FIG. 17. A fluorescent micrograph showing the reticularlocalization pattern of rosGFP1 expressed in the mitochondrial matrix ofan in vitro cultured HeLa cell, via fusion at the DNA level to themitochondrial targeting sequence of the E₁α subunit of pyruvatedehydrogenase.

[0037]FIG. 18. Response of rosGFP1 to H₂O₂ and DTT induced redoxpotential changes in HeLa cell mitochondria. H₂O₂ and DTT were added atthe indicted time points to a final concentration of 1 mM and 30 mM,respectively. The Fluorescence Intensity axis corresponds to theindividual wavelengths, whereas the Ratio 400/480 axis corresponds tothe ratio of the two wavelength channels.

[0038]FIG. 19. NADH-dependent reduction of rosGFP1 via lipoamidedehydrogenase. Each bar represents the percent reduction of oxidizedrosGFP1 by 1-2 μL LDH, 1 mM lipoate, 1 mM NADH, and/or 1 mM DTT. Sampleswere equilibrated at 22° C. for one hour after which the fluorescenceexcitation was scanned from 325 to 525 nm. Percent reduction values weredetermined by the fluorescence at 490 nm with 100% corresponding toreduction by DTT.

[0039]FIG. 20. Fluorescence excitation spectra of rosGFP2 at varyingconcentrations of DTT_(red) and DTT_(ox). Fluorescence emissionintensity was monitored at 510 nm and normalized to the maximumintensity of the fully reduced spectrum (solid line).

[0040]FIG. 21. Redox equilibrium titration of rosGFP2 withdithiothreitol. The relative amount of reduced rosGFP2 at equilibrium(R) was measured using a ratio of the rosGFP2 fluorescence at 510 nm(excitation 490:425 nm). Oxidized rosGFP2 (1 μM) was incubated for fourhours in 75 mM HEPES (pH 7.0), 140 mM NaCl, and 1 mM EDTA, containingvarying ratios of DTT_(red) to DTT_(ox) (1 mM total). The equilibriumconstant was determined by fitting the data according to equation 3.After nonlinear regression, a K_(eq) of 2.05×10⁻² was obtained(correlation coefficient: 0.998).

SEQUENCE LISTING

[0041] The nucleic and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three letter code for amino acids, as defined in37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown,but the complementary stand is understood as included by any referenceto the displayed strand. In the accompanying sequence listing:

[0042] SEQ ID NO: 1 shows the amino acid sequence of wild-type GFP.

[0043] SEQ ID NO: 2 shows the nucleic acid and amino acid sequence ofrosGFP2.

[0044] SEQ ID NO: 3 shows the amino acid sequence of rosGFP2.

[0045] SEQ ID NO: 4 shows the nucleic acid and amino acid sequence ofrosGFP1.

[0046] SEQ ID NO: 5 shows the amino acid sequence of rosGFP1.

[0047] SEQ ID NO: 6 shows the nucleic acid and amino acid sequence ofrosGFP4.

[0048] SEQ ID NO: 7 shows the amino acid sequence of rosGFP4.

[0049] SEQ ID NO: 8 shows the nucleic acid and amino acid sequence ofrosGFP3.

[0050] SEQ ID NO: 9 shows the amino acid sequence of rosGFP3.

[0051] SEQ ID NO: 10 shows the nucleic acid and amino acid sequence ofrosGFP6.

[0052] SEQ ID NO: 11 shows the amino acid sequence of rosGFP6.

[0053] SEQ ID NO: 12 shows the nucleic acid and amino acid sequence ofrosGFP5.

[0054] SEQ ID NO: 13 shows the amino acid sequence of rosGFP5.

DETAILED DESCRIPTION

[0055] I. Abbreviations GFP green fluorescent protein rosGFPredox-sensitive GFP WtGFP wild-type GFP

[0056] II. Terms

[0057] Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

[0058] In order to facilitate review of the various embodiments, thefollowing explanations of specific terms are provided:

[0059] Animal: Living multi-cellular vertebrate organisms, a categorythat includes, for example, mammals and birds. The term mammal includesboth human and non-human mammals. Similarly, the term “subject” includesboth human and veterinary subjects.

[0060] Antibody: A polypeptide substantially encoded by animmunoglobulin gene or imunoglobulin genes, or fragments thereof, whichspecifically binds and recognizes an analyte (antigen). Immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as the myriad immunoglobulin variableregion genes.

[0061] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. For instance, FAbs, Fvs, and single-chain Fvs (SCFvs) thatbind to GFP would be GFP-specific binding agents. Antibody fragments aredefined as follows: (1) Fab, the fragment which contains a monovalentantigen-binding fragment of an antibody molecule produced by digestionof whole antibody with the enzyme papain to yield an intact light chainand a portion of one heavy chain; (2) Fab′, the fragment of an antibodymolecule obtained by treating whole antibody with pepsin, followed byreduction, to yield an intact light chain and a portion of the heavychain; two Fab′ fragments are obtained per antibody molecule; (3)(Fab″)2, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)2, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. The term “antibody,” as used herein, also includes antibodyfragments either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA methodologies.

[0062] cDNA (complementary DNA): A piece of DNA lacking internal,non-coding segments (interns) and transcriptional regulatory sequences.cDNA may also contain untranslated regions (UTRs) that are responsiblefor translational control in the corresponding RNA molecule. cDNA isusually synthesized in the laboratory by reverse transcription frommessenger RNA extracted from cells.

[0063] Conservative variations: Variants of a particular nucleic acidsequence, which encode identical or essentially identical amino acidsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given polypeptide.For instance, the codons CGU, CGC, CGA, COG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified within a protein encoding sequence, the codon can be alteredto any of the corresponding codons described without altering theencoded protein. Such nucleic acid variations are “silent variations,”which are one species of conservative variations. Each nucleic acidsequence herein that encodes a polypeptide also describes every possiblesilent variation. The genetic code is shown in Table 1. TABLE 1 Firstposition Second position Third position (5′ end) U C A G (3′ end) U PheSer Tyr Cys U Phe Ser Tyr Cys C Leu Ser Stop Stop A Leu Ser Stop Trp G CLeu Pro His Arg U Leu Pro His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg GA Ile Thr Asn Ser U Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr Lys ArgG G Val Ala Asp Gly U Val Ala Asp Gly C Val Ala Glu Gly A Val Ala GluGly G

[0064] One of skill will recognize that each codon in a nucleic acid(except AUG, which is ordinarily the only codon for methionine) can bemodified to yield a functionally identical molecule by standardtechniques. Accordingly, each “silent variation” of a nucleic acid thatencodes a polypeptide is implicit in each described sequence.

[0065] Furthermore, one of ordinary skill will recognize that individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (for instanceless than 5%, in some embodiments less than 1%) in an encoded sequenceare conservative variations where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.

[0066] Conservative amino acid substitutions providing functionallysimilar amino acids are well known in the art. The following six groupseach contain amino acids that are conservative substitutions for oneanother:

[0067] 1) Alanine (A), Serine (S), Threonine (T);

[0068] 2) Aspartic acid (D), Glutamic acid (E);

[0069] 3) Asparagine (N), Glutaminc (Q);

[0070] 4) Arginine (R), Lysine (K);

[0071] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0072] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Not allresidue positions within a protein will tolerate an otherwise“conservative” substitution. For instance, if an amino acid residue isessential for a function of the protein, even an otherwise conservativesubstitution may disrupt that activity. By way of example, in a GFP theresidues that compose the chromophore do not generally tolerate aminoacid substitutions.

[0073] Epitope tags: Short stretches of amino acids to which a specificantibody can be raised, which in some embodiments allows one tospecifically identify and track the tagged protein that has been addedto a living organism or to cultured cells. Detection of the taggedmolecule can be achieved using a number of different techniques.Examples of such techniques include: immunohistochemistry,immunoprecipitation, flow cytometry, immunofluorescence microscopy,ELISA, immunoblotting (“western”), and affinity chromatography. Examplesof useful epitope tags include FLAG, T7, HA (hemagglutinin) and myc.

[0074] Expression control sequence: This phrase refers to nucleotidesequences that regulate the expression of a nucleotide sequence to whichthey are operatively linked. Expression control sequences are“operatively linked” to a nucleotide sequence when the expressioncontrol sequences control and regulate the transcription and, asappropriate, translation of the nucleotide sequence. Thus, expressioncontrol sequence(s) can include promoters, enhancers, transcriptionterminators, a start codon (i.e, ATG) in front of a protein-encodingsequence, intron splicing signals, and stop codons.

[0075] Fluorescent property: A characteristic of a fluorescent molecule.Examples of fluorescent properties include the molar extinctioncoefficient at an appropriate excitation wavelength, the fluorescencequantum efficiency, the shape of the excitation spectrum or emissionspectrum (the “fluorescence spectrum”, the excitation wavelength maximumand emission wavelength maximum, the ratio of excitation amplitudes attwo different wavelengths, the ratio of emission amplitudes at twodifferent wavelengths, the excited state lifetime, or the fluorescenceanisotropy. A measurable difference in any one of these propertiesbetween wild-type Aequorea GFP and the mutant form is useful. Ameasurable difference can be determined by determining the amount of anyquantitative fluorescent property, e.g., the amount of fluorescence at aparticular wavelength, or the integral of fluorescence over the emissionspectrum. Determining ratios of excitation amplitude or emissionamplitude at two different wavelengths (“excitation amplitude ratioing”and “emission amplitude ratioing,” respectively) for a particularmolecule are advantageous. The ratioing process provides an internalreference and cancels out variations, for instance in the absolutebrightness of the excitation source, the sensitivity of the detector,and light scattering or quenching by the sample.

[0076] Fusion protein: Proteins that have two (or more) parts fusedtogether, which are not found joined together in nature. In general, thetwo domains are genetically fused together, in that nucleic acidmolecules that encode each protein domain are functionally linkedtogether, for instance by a linker oligonucleotide, thereby producing asingle fusion-encoding nucleic acid molecule. The translated product ofsuch a fusion-encoding nucleic acid molecule is the fusion protein.

[0077] Green fluorescent protein (GFP): GFP is a 238 amino acid,spontaneously fluorescent protein, originally isolated from the PacificNorthwest jellyfish Aequorea victoria. The amino acid sequence of wtGFPis shown in SEQ ID NO: 1. Tis protein has become an extremely populartool in molecular and call biology (for reviews: Tsien, Annu. Rev.Biochem. 67:509-544, 1998; Remington, In Bioluminescence andchemiluminescence (eds. T. O. Baldwin and M. M. Sigler), pp. 195-211,2000, Academic, San Diego, Calif.). Originally GFP was used as a passiveindicator of gene expression and protein localization. More recently,GFP has taken on the role of an active indicator of such things asintracellular H⁺, Ca²⁺, and halide ion concentrations (Kneen et at.,Biophys. J. 74:1591-1599, 1998; Llopis et al., Proc. Natl. Acad Sci. USA95:6803-6808, 1998; Baird et al., Proc. Natl. Acad. Sci. USA96:11241-11246, 1999; Jayaraman et a., J. Biol. Chem.275:6047-6050,2000).

[0078] In addition to GFP being highly fluorescent, protease resistant,and very stable throughout a wide range of pH and solvent conditions, italso has the advantage of being functional as a single protein encodedby a single gene. These traits result in a biological probe moleculethat can be expressed in nearly all organisms. It also can be targetedto subcellular organelles by a host cell, for instance through theinclusion of a targeting sequence on the construction from which it isexpressed. GFP is a non-invasive indicator, which allows for experimentsto be conducted and monitored in a single cell over a period of time.

[0079] GFPs as discussed herein (including rosGFPs) can be expressed asfusion proteins. The GFP protein can be functionally fused to, forinstance, a tag (such as an epitope tag), a targeting molecule (such asa targeting peptide), or a protein (or fragment thereof) that providesan additional function, such as a biochemical, biological, orlocalization function. The construction and production of fusionproteins is well known to one of ordinary skill in the art.

[0080] A “mutant” GFP is a green fluorescent protein (or nucleic acidencoding such) that has at least one residue that is different from(mutated from) the wtGFP. Mutations include, for instance, conservativeor non-conservative amino acid substitutions, silent mutations (whereinthe nucleic acid sequence is different from wild-type at a particularresidue, but the amino acid sequence is not), insertions (includingfusion proteins), and deletions. Myriad mutant GFPs are known, includingfor instance those disclosed in the following patent documents: U.S.Pat. Nos. 5,804,387; 6,090,919; 6,096,865; 6,054,321; 5,625,049;5,874,304; 5,777,079; 5,968,750; 6,020,192; and 6,146,826; and publishedinternational patent application WO 99/64592.

[0081] Specific examples of mutant GFPs include proteins in which thefluorescence spectrum of the mutant is responsive to an environmentalvariable, such as temperature, proton concentration (pH), saltconcentration, and redox status. Particular mutant GFPs as providedherein are sensitive to redox status, and others are responsive to bothredox status and pH. A fluorescence spectrum is “responsive” to anenvironmental variable if the spectrum changes with changes in thatvariable.

[0082] Immunoassay: An assay that utilizes an antibody to specificallybind an analyte. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, detect,and/or quantify the analyte, or alternately using a particularly analyte(e.g., an antigen) to isolate, target, detect, and/or quantify theantibody.

[0083] In vitro amplification: Techniques that increases the number ofcopies of a nucleic acid molecule in a sample or specimen. An example ofamplification is the polymerase chain reaction, in which a biologicalsample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to nucleic acid template in the sample. Theprimers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of in vitroamplification may be characterized by electrophoresis, restrictionendonuclease cleavage patterns, oligonucleotide hybridization orligation, and/or nucleic acid sequencing, using standard techniques.Other examples of in vitro amplification techniques include stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see WO 90/01069);ligase chain reaction amplification (see EP-A-320 308); gap fillingligase chain reaction amplification (see U.S. Pat. No. 5,427,930);coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); andNASBA™ RNA transcription-free amplification (see U.S. Pat. No.6,025,134).

[0084] Isolated: An “isolated” biological component (such as a nucleicacid molecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

[0085] Label: A composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³²P (or other radioisotope), fluorescent dyes,fluorescent proteins, electron-dense reagents, enzymes (e.g., for use inan ELISA), biotin, dioxigenin, or haptens and proteins or peptides forwhich antisera or monoclonal antibodies are available. A label oftengenerates a measurable signal, such as radioactivity, fluorescent lightor enzyme activity, which can be used to detect and/or quantitate theamount of labeled molecule.

[0086] Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer ineither single-or double-stranded form. Unless otherwise limited, thisterm encompasses known analogs of natural nucleotides that can functionin a similar manner as naturally occurring nucleotides. When a nucleicacid molecule is represented herein by a DNA sequence, the correspondingRNA molecules are likewise understood, in which “U” replaces “T.”

[0087] Oligonucleotide: An oligonucleotide is a plurality of joinednucleotides joined by native phosphodiester bonds, between about 6 andabout 300 nucleotides in length. An oligonucleotide analog refers tomoieties that function similarly to oligonucleotides but havenon-naturally occurring portions. For example, oligonucleotide analogscan contain non-naturally occurring portions, such as altered sugarmoieties or inter-sugar linkages, such as a phosphorothioateoligodeoxynucleotide. Functional analogs of naturally occurringpolynucleotides can bind to RNA or DNA, and include peptide nucleic acid(PNA) molecules.

[0088] Particular oligonucleotides and oligonucleotide analogs caninclude linear sequences up to about 200 nucleotides in length, forexample a sequence (such as DNA or RNA) that is at least 6 bases, forexample at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200bases long, or from about 6 to about 50 bases, for example about 10-25bases, such as 12, 15 or 20 bases.

[0089] Open reading frame: A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

[0090] Operably linked: A first nucleic acid sequence is operably linkedwith a second nucleic acid sequence when the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame. Similarly, two peptide or polypeptide sequences areconsidered to be operably linked if they are linked to each other insuch a way that they function in the intended manner.

[0091] Polypeptide or Protein: A polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The term “recombinant protein” refers to a protein that isproduced by expression of a nucleotide sequence (which encodes theprotein) from a recombinant DNA molecule.

[0092] Preferred mammalian codon(s): The subset of codons from among theset of all possible codons encoding an amino acid that are mostfrequently used in proteins expressed in mammalian cells. Table 2summarizes the preferred mammalian codons for each amino acid: TABLE 2Amino Acid Preferred codons* Gly GGC, GGG Glu GAG Asp GAC Val GUG, GUCAla GCC, GCU Ser AGC, UCC Lys AAG Asn AAC Met AUG Ile AUC Thr ACC TrpUGG Cys UGC Tyr UAU, UAC Leu CUG Phe UUC Arg CGC, AGG, AGA Gln CAG HisCAC Pro CCC

[0093] Primers: Primers are short nucleic acid molecules, for instanceDNA oligonucleotides 10 nucleotides or more in length. Longer DNAoligonucleotides May be about 15, 20, 25, 30 or 50 nucleotides or morein length. Primers can be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand, and then the primer extended along the target DNAstrand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other nucleic-acid amplification methods known in theart

[0094] Methods for preparing and using nucleic acid primers aredescribed, for example, in Sambrook et al. (In Molecular Cloning: ALaboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed) (InCurrent Protocols in Molecular Biology, John Wiley & Sons, New York,1998), and Innis et al. (PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc., San Diego, Calif., 1990).Amplification primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5, ©1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.). One of ordinary skill in the art will appreciate thatthe specificity of a particular probe or primer increases with itslength.

[0095] Probes: A probe comprises an isolated nucleic acid attached to adetectable label or other reporter molecule. Typical labels includeradioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent or fluorescent agents, haptens, and enzymes. Methodsfor labeling and guidance in the choice of labels appropriate forvarious purposes are discussed, e.g., Sambrook et al. (In MolecularCloning: A Laboratory Manual, CSHL, New York:, 1989) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,1998).

[0096] Promoter: A promoter is an ordered set of nucleic acid controlsequences that direct transcription of a nucleic acid. A promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements that can be located as much as several thousand basepairs from the start site of transcription.

[0097] Purified: The term “purified” does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiedprotein preparation is one in which the protein referred to is more purethan the protein in its natural environment within a cell or within aproduction reaction chamber (as appropriate).

[0098] Recombinant: A recombinant nucleic acid is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two otherwise separated segments ofsequence. This artificial combination can be accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques.

[0099] Recombinant host cell: A cell (such as a bacterial, plant, oranimal cell) that comprises a recombinant nucleic acid molecule. Thus,for example, recombinant host cells can express genes and/or proteinsthat are not found within the native (non-recombinant) form of the cell.

[0100] Redox status: A measurement of the oxidation-reduction (redox)potential of an environment, for instance the environment within a cellor a subcellular compartment

[0101] Fundamentally, redox reactions are a family of reactions that areconcerned with the transfer of electrons between species. Oxidationrepresents a loss of electrons, reduction a gain of electrons.Oxidation-reduction reactions always occur together, and the electronsgained by the molecule that is reduced must balance those given up bythe substance that is oxidized. The oxidation-reduction potential (orredox status) of a solution is a measurement of the oxidation orreduction force of the solution, and is indicative of the oxidation orreduction ability.

[0102] Redox status of any solution can be measured. For instance, theredox status of the solution within a cell (i.e., the cytosol) can beanalyzed using the provided rosGFPs. Similarly, the redox status of thesolution within a subcellular organelle (such as the nucleus,mitochondria, plastid, vacuole, secretory pathway compartment and soforth) can be analyzed

[0103] Stringent conditions: A set of temperature and ionic conditionsused in a nucleic acid hybridization. Stringent conditions are sequencedependent and are different under different environmental parameters.Generally, stringent conditions are selected to be about 5° C. to 20° C.lower than the thermal melting point (T_(m)) for the specific targetsequence. The T_(m) is the temperature (under defined ionic strength andpH) at which 50% of the target sequence hybridizes to a perfectlymatched (100% identical) probe molecule.

[0104] Substantially identical/similar: An amino acid sequence or anucleotide sequence is substantially identical (or substantiallysimilar) to a reference sequence if the amino acid sequence ornucleotide sequence has at least 80% sequence identity with thereference sequence over a given window of comparison. Thus,substantially similar sequences include those having, for example, atleast 85% sequence identity, at least 90% sequence identity, at least95% sequence identity or at least 99% sequence identity. Two sequencesthat are 100% identical to each other are, of course, also substantiallyidentical.

[0105] Transformed: A transformed cell is a cell into which has beenintroduced a nucleic acid molecule by molecular biology techniques. Asused herein, the term transformation encompasses all techniques by whicha nucleic acid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

[0106] Vector: A nucleic acid molecule as introduced into a host cell,thereby producing a transformed host cell. A vector may include nucleicacid sequences that permit it to replicate in a host cell, such as anorigin of replication. A vector may also include one or more selectablemarker genes and other genetic elements known in the art.

[0107] Unless otherwise explained, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. The singularterms “a,” “an,” and “the” include plural referents unless contextclearly indicates otherwise. Comprises means includes. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples am illustrative only andnot intended to be limiting.

[0108] III. Redox-Senstive Green Fluorescent Proteins

[0109] Many fundamental biological processes rely upon a properlymaintained intracellular redox environment (Cuozzo and Kaiser, Nat.CellBiol. 1:130- 135, 1999; Debarbieux and Beckwith, Cell 99:117-119, 1999;Hall, Eur. J. Clin. Invest. 29:238-245, 1999; Cai and Jones, J.Bioenerg. Biomemb. 31:327-334, 1999). Moreover, reactive oxygen specieslike O₂—⁻; HO—, or H₂O_(2,) arise in cells by a variety of processesincluding light, radiation, or the respiratory chain. These radicalspecies present major threats to organisms by damaging DNA, membranes,or other cellular components.

[0110] The importance of redox status to biological process might implythat researchers have numerous well-established techniques formonitoring redox potentials in vivo. However, this is not the case.Current redox-sensing methods are invasive, require large sample sizes,are labor intensive, and do not allow for real-time determinations onliving cells.

[0111] This disclosure describes a new class of GFP variants (rosGFPs)that display ratiometric excitation properties as a function of redoxpotential. rosGFP biosensors allow for real-time measurements of redoxstatus on small groups of cells, individual cells, or even withincertain cellular organelles. As a result of being genetically encoded,these molecules are non-invasive, and their fluorescence characteristicsthey are easy to use as sensing molecules.

[0112] To create a redox sensor that overcomes many of the drawbacks ofother methods of in vivo redox status determination, site-directedmutagenesis was carried out on the green fluorescent protein (GFP). Bymutating residues near the chromophore in CFP to cysteines, novelredox-sensitive GFPs (rosGFPs) were constructed. They displayratiometric dual-excitation fluorescent properties as a function ofredox state, with apparent redox potentials of −0.272 to −0.299 V.

[0113] Unexpectedly, these rosGFPs also exhibited ratiometricdual-emission properties in response to pH changes, providing the uniquepossibility to simultaneously monitor redox potential and pH changeswith the same probe. T65S reversion leads to pH-independent redoxsensors with altered redox potentials and large UV excitation peaks,which can be used to overcome background levels of cellularauto-fluorescence.

[0114] Crystal structure analyses of an oxidized and reduced rosGFP to1.9 and 2.0 Å, respectively, indicate that changes in the structure onreduction or oxidation of the disulfide bridge could account for theobserved spectral changes.

[0115] As shown herein using cultured HeLa cells, rosGFPs can reversiblyrespond to exogenous redox stimuli and have redox potentials near thatof mitochondria, with the apparent redox potential of mitochondriaestimated to more reducing than about −0.30 V. The apparent redoxpotential of the cytosol also was found to be more reducing than −0.30V.

[0116] One example redox sensitive GFP mutant (rosGFP2) differs fromwild-type GFP in that it contains the following amino acid residuemutations: C48S/S65T/Q80R/S147C/Q204C (SEQ ID NOs: 2 and 3). FIG. 1illustrates how the fluorescence of rosGFP2 varies in response tochanges in redox potential. The spectra show two excitation peaks, onenear 400 nm and the other at about 490 mm, with a clear isosbestic pointseparating them. These dual excitation peaks respond in oppositedirections to redox potential, making this indicator ratiometric forredox potential. Ratiometric indicators are known to reduce or eliminatedistortions of data caused by photobleaching, indicator concentration,variable cell thickness, illumination stability, excitation pathlength,and non-uniform indicator distribution within cells or between groups ofcells (Grynkiewicz et al., J. Biol. Chem. 260:3440-3450, 1985).

[0117] By monitoring the chromophore fluorescence at the 490 nmexcitation peak as a function of redox potential, the apparent redoxpotential of rosGFP2 was found FIG. 2 shows the titration of rosGFP2with dithiothreitol. From this plot the apparent redox potential is−0.279 volts. To demonstrate that the redox-sensitive probe responds toin vivo redox changes, it was targeted to the mitochondria of HeLacells. As seen in FIG. 3, the starting ratio of the 400 to 490 nm peakin mitochondria is around 0.28. After the addition of the redox-cyclingagent vitamin K₃, the ratio dramatically increases to about 0.42. Thisincrease in the ratio is indicative of an oxidation event that is to beexpected according to the reaction catalyzed by vitamin K₃ (Suttie,Annu. Rev. Biochem. 54:459-477, 1985). After 30 total minutes, theaddition of dithiothreitol elicits the opposite response as indicated bythe smaller ratio.

[0118] Characteristics of several redox-sensitive GFP mutants aresummarized in Table 3. TABLE 3 Mutant Excitation Redox nameSubstitutions Peaks Potential* rosGFP2 C48S/S65T/S147C/Q204C 400 nm−0.272 (SEQ ID NOs: 2 and 3) 490 nm rosGFP1 C48S/S147C/Q204C 397 nm−0.288 (SEQ ID NOs: 4 and 5) 477 nm rosGFP4 C48S/S65T/N149C/S202C 400 nm−0.286 (SEQ ID NOs: 6 and 7) 490 nm rosGFP3 C48S/N149C/S202C 392 nm−0.299 (SEQ ID NOs: 8 and 9) 475 nm rosGFP6C48S/S65T/S147C/N149C/S202C/Q204C 398 nm −0.280 (SEQ ID NOs: 10 and 11)490 nm rosGFP5 C48S/S147C/N149C/S202C/Q204C 395 nm −0.296 (SEQ ID NOs:12 and 13) 475 nm

[0119] Properties of rosGFPs

[0120] Examples of redox-sensitive GFPs described herein have manydesirable qualities. In addition to being genetically encodedfluorescent indicators, they are ratiometric. This ratiometric behaviorhas the advantage of reducing or eliminating distortions of data causedby photobleaching, indicator concentration, variable cell thickness,illumination stability, excitation path length, and non-uniformindicator distribution within cells or between groups of cells(Grynkiewicz et al., J. Biol. Chem. 260:3440-3448, 1985; Whitaker etal., Anal. Biochem. 194:330-344, 1991). In addition, a ratio ofexcitation wavelengths greatly minimizes the contribution of pHartifacts to the fluorescent signal over a pH range from 6 to 8.5.Similarly, a ratio of emission wavelengths can result in pH monitoringwithout artifacts introduced by the redox state of the biosensor.

[0121] The second-generation rosGFPs, with more closely matchedexcitation peak amplitudes, may aid in fluorescence microscopyexperiments by allowing the same camera/detector settings to captureboth images that constitute the ratio. Furthermore, second-generationrosGFPs have a larger 400 nm fluorescence amplitude, which is desirablefor detection of the rosGFP probe over background levels of cellularautofluorescence.

[0122] Accuracy of Standard Redox Potentials

[0123] The main problem in the determination of redox potentials is thatE_(o) values cannot be measured directly, but rather are calculated fromthe known E_(o), of another redox couple, equilibrated with the redoxcouple of unknown E_(o). The standard redox potential can thus vary overa large range depending on experimental conditions and the choice ofredox couple.

[0124] In this disclosure, the value of −0.323 V for the dithiothreitolcouple was chosen, because it has been very accurately determined in0.05-0.02 M phosphate buffer at pH 7.0 and 30.0°+/−0.5° C. using thelipoamide-lipoamide dehydrogenase couple (Szajewski and Whitesides, J.Am. Chem. Soc. 102:2011-2026, 1980). Because redox potentials are pH andtemperature dependent, all redox equilibria were measured at the same pH(7.0) and temperature (30° C.) as was used to evaluate the standardredox potential of the dithiothreitol couple. Another reason to use thevalue of −0.323 V for the dithiothreitol couple is that the standardpotentials of other commonly used thiol reagents (including the GSH-GSSGcouple) have been determined under identical conditions (Szajewski andWhitesides, J. Am. Chem. Soc. 102:2011-2026, 1980). This will allow forthe direct comparison of redox potentials determined by use of rosGFPsto those determined by other methods, provided the experimentalconditions are reproduced.

[0125] For the DTT_(red)-DTT_(ox) couple, the standard potentialthroughout literature is well accepted to be about −0.330 V at nearbiochemist's standard state of pH 7 and 25° C. However, such agreementis not always the norm. In the case of the glutathione redox couple,very different values for the standard redox potential have beenreported. The published values deviate from a somewhat oxidizing valueof −0.205 V to a more reducing value of −0.250 V (Szajewski andWhitesides, J. Am. Chem. Soc. 102:2011-2026, 1980; Rost and Rapoport,Nature 201:185, 1964; Torchinsky, Sulfur in proteins. Pergamon PressLtd., 1981, New York, N.Y.). The 45 mV difference in literature valuesmight be problematic when trying to compare redox potentials of rosGFPsto previously determined estimates of the redox potential inside cells.

[0126] Even with the large discrepancy in the standard potential ofGSH-GSSG, results reported herein indicate that in vivo redox potentialsmay be morn reducing than previous estimates. Hwang et al., using thetetrapeptide N-Acetyl-Asn-Thr-Cys-NH₂ to measure the ratio of thiol todisulfide in the cytosol and secretory pathway of cultured cells,concluded that the cytosol is more reducing than the secretory pathwaywith an approximate redox potential of −0.221 to −0.236 V versus −0.170to −0.185 V, respectively (Hwang et al., Science 257:1496-1502, 1992).In addition, based on prior determinations of the concentration andratio of GSH to GSSG in mitochondria, the redox potential of thiscompartment is calculated to be −0.210 to −0.236 V. However, Keese etat. recently developed an indicator of redox state in which theytransfer glutathione reductase crystals into the cytosol of living cellsand then detect a color change in the crystals (Keese et al., FEBS Lett.447:135-138, 1999). Using that method, Keese et al. determined the redoxpotential of the cytosol of human fibroblasts to be more reducing than−0.270 V at pH 7.4 and 37° C. That result is in agreement with theestimate reported herein of the cytosolic redox potential for HeLa cellsbeing more negative than −0.330 V.

[0127] IV. Construction of rosGFPs

[0128] The examples below provide specific methods for producing certainembodiment rosGFPs. More generally, in light of the disclosure hereinthat disulfide bonding pairs can be incorporated into GFP to produceredox-sensitive GEP mutants, it will be understood to one of ordinaryskill in the art that many different methods could be used to makerosGFPs.

[0129] A. GFP Nucleic Acids and Site-Directed Mutations

[0130] DNA encoding wtGFP is available commercially, for example fromCLONTECH (Palo Alto, Calif.). Methods of producing mutants containing apredetermined nucleotide sequence are well known to those of ordinaryskill in the art. Two widely used methods are Kunkel mutagenesis and PCRmutagenesis. A detailed description of the two techniques can be foundin Current Protocols in Molecular Biology, Wiley Interscience, 1987,Sections 8.1 and 8.5 respectively. See also Kunkel, Proc. Acad. Nat.Sci. USA 82:488-492, 1985; and Saiki et al., Science 239:487-491, 1988.

[0131] It is also possible to synthesize mutant GFPs and DNA encodingfor mutant GFPs directly, by synthetic methods well known in the art.The mutant sequences can be expressed in a variety of systems, includingbacterial, yeast, plant and mammalian cells. DNA encoding the mutant GFPis inserted in an expression vector and transformed into cells ofinterest The sequence encoding for GFP is inserted in the vector in thecorrect reading frame near a strong promoter, and the expression of GFPis induced. Vectors suitable for specific expression systems are wellknown in the art, and are widely available commercially. Vectors havingcodons opted for expression in a variety of systems, including yeast andmammalian cells, are also available. For a description of silentnucleotide sequence mutations optimized for mammalian expression, seefor example Haas et al. (Curr. Biol. 6:315-324, 1996).

[0132] B. Expression of rosGFPs

[0133] One skilled in the art will understand that there are myriad waysto express a recombinant protein such that it can subsequently bepurified. In general, an expression vector carrying the nucleic acidsequence that encodes the desired protein will be transformed into amicroorganism for expression. Such microorganisms can be prokaryotic(bacteria) or eukaryotic (g., yeast). One appropriate species ofbacteria is Escherichia coli (E. coli), which has been used extensivelyas a laboratory experimental expression system. A eukaryotic expressionsystem will be preferred where the protein of interest (or a domainwithin a fusion protein of interest) requires eukaryote-specificpost-translational modifications such as glycosylation. Also, proteincan be expressed using a viral (e.g., vaccinia) based expression system.Protein can also be expressed in animal cell tissue culture, and such asystem will be appropriate where animal-specific protein modificationsare desirable or required in the recombinant protein.

[0134] The expression vector can include a sequence encoding a targetingpeptide, positioned in such a way as to be fused to the coding sequenceof the rosGFP. Targeting domains may allow the fusion protein to betargeted to specific extra-cellular locations, or simply to be secretedfrom the cell, and may be removed during or soon after synthesis of thefusion protein. In addition, multiple targeting peptides can be includedin a single fusion, for instance a peptide/domain that directs thefusion protein to be secreted, and another peptide/domain that directsthe secreted protein to a target (cell, tissue, organ, etc.). Variousappropriate prokaryotic and eukaryotic targeting peptides, and nucleicacid molecules encoding such, are known to one of ordinary skill in theart Through the use of a eukaryotic secretion-type signal sequence, arosGFP fusion protein can be expressed in a transgenic animal (forinstance a cow, pig, or sheep) in such a manner that the fusion proteinis secreted into the milk of the animal. Targeting protein portions alsomay be used to ensure that a transgenically expressed fusion protein issecreted into the circulatory system of the transgenic animal, therebypermitting the fusion protein to be transported to a target (cell,tissue, organ, etc.).

[0135] Optional localization peptide sequences may be, for instance, anuclear localization sequence, an endoplasmic reticulum localizationsequence, a peroxisome localization sequence, a mitochondriallocalization sequence, or a localized protein. Localization sequencescan be targeting sequences which are described, for example, in “ProteinTargeting,” chapter 35 of Stryer, Biochemistry (4th ed.). W. H. Freeman,1995. The localization sequence can also be a localized protein (ordomain thereof). Some localization sequences include the followingnon-limiting examples (Table 4): TABLE 4 Subcellular locationLocalization sequence(s) nucleus KKKRK mitochondrionMLRTSSLFTRRVQPSLFRNILRLQST-* endoplasmic reticulum KDEL** (providingretention in the ER) when used with a signal sequence* peroxisome SKF**prenylation or insertion into CaaX, CC, CXC, or CCXX** plasma membranecytoplasmic side of plasma fusion to SNAP-25 membrane Golgi fusion tofurin

[0136] Vectors suitable for stable transformation of culturable cellsare also well known. Typically, such vectors include a multiple-cloningsite suitable for inserting a cloned nucleic acid molecule, such that itwill be under the transcriptional control of 5′ and 3′ regulatorysequences. In addition, transformation vectors include one or moreselectable markers; for bacterial transformation this is often anantibiotic resistance gene. Such transformation vectors typically alsocontain a promoter regulatory region (e.g., a regulatory regioncontrolling inducible or constitutive expression), a transcriptioninitiation start site, a ribosome binding site, an RNA processingsignal, and a transcription termination site, each functionally arrangedin relation to the multiple-cloning site. For production of largeamounts of recombinant proteins, an inducible promoter is preferred.This permits selective production of the recombinant protein, and allowsboth higher levels of production than constitutive promoters, andenables the production of recombinant proteins that may be toxic to theexpressing cell if expressed constitutively.

[0137] In addition to these general guidelines, proteinexpression/purification kits are produced commercially. See, forinstance, the QIAexpress™ expression system from QIAGEN (Chatsworth,Calif.) and various expression systems provided by INVITROGEN (Carlsbad,Calif.).

[0138] C. Purification of rosGFPs

[0139] One skilled in the art will understand that there are myriad waysto purify recombinant polypeptides, and such typical methods of proteinpurification may be used to purify the disclosed rosGFPs. Such methodsinclude, for instance, protein chromatographic methods including ionexchange, gel filtration, HPLC, monoclonal antibody affinitychromatography and isolation of insoluble protein inclusion bodies afterover production. In addition, purification affinity-tags, for instance asix-histidine sequence, may be recombinantly fused to the protein andused to facilitate polypeptide purification (e.g., in addition toanother functionalizing portion of the fusion, such as a targetingdomain or another tag). A specific proteolytic site, for instance athrombin-specific digestion site, can be engineered into the proteinbetween the tag and the remainder of the fusion to facilitate removal ofthe tag after purification, if such removal is desired.

[0140] Commercially produced protein expression/purification kitsprovide tailored protocols for the purification of proteins made usingeach system. See, for instance, the QIAexpress™ expression system fromQIAGEN (Chatsworth, Calif.) and various expression systems provided byINVITROGEN (Carlsbad, Calif.). Where a commercial kit is employed toproduce a rosGFP, the manufacturer's purification protocol is apreferred protocol for purification of that protein. For instance,proteins expressed with an amino-terminal hexa-histidine tag can bepurified by binding to nickel-nitilotriacetic acid (Ni-NTA) metalaffinity chromatography matrix (The QLAexpressionist, QIAGEN, 1997).

[0141] D. Optional Modifications to rosGFPs

[0142] Optionally, redox-sensitive GFP variants/mutants can be“humanized” as described in U.S. Pat. No. 5,874,304, or can containother mutations (such as substitutions) as dictated by the end-use ofthe protein.

[0143] Also, the mutant GFP proteins can be expressed as part of afusion protein. The construction of fusion proteins from domains ofknown proteins, or from whole proteins or proteins and peptides, is wellknown. In general, a nucleic acid molecule that encodes the desiredprotein and/or peptide portions are joined using genetic engineeringtechniques to create a single, operably linked fusion oligonucleotide.Appropriate molecular biological techniques may be found in Sambrook etal. (1989). Examples of genetically engineered multi-domain proteins,including those joined by various linkers, and those containing peptidetags, can be found in the following patent documents:

[0144] U.S. Pat. No. 5,994,104 (“Interleukin-12 fusion protein”);

[0145] U.S. Pat. No. 5,981,177 (“Protein fusion method andconstruction”);

[0146] U.S. Pat. No. 5,914,254 (“Expression of fusion polypeptidestransported out of the cytoplasm without leader sequences”);

[0147] U.S. Pat. No. 5,856,456 (“Linker for linked fusionpolypeptides”);

[0148] U.S. Pat. No. 5,767,260 (“Antigen-binding fusion proteins”);

[0149] U.S. Pat. No. 5,696,237 (“Recombinant antibody-toxin fusionprotein”);

[0150] U.S. Pat. No. 5,587,455 (“Cytotoxic agent against specific virusinfection”);

[0151] U.S. Pat. No. 4,851,341 (“lmmunoaffinity purification system”);

[0152] U.S. Pat. No. 4,703,004 (“Synthesis of protein with anidentification peptide”); and

[0153] WO 98/36087 (“Immunological tolerance to HIV epitopes”).

[0154] In particular, patent disclosures related to fusion proteinscontaining a GFP moiety include the following:

[0155] U.S. Pat. No. 6,180,343 (“Green fluorescent protein fusions withrandom peptides”);

[0156] WO 99/54348 (“Rapidly degrading GFP-fusion proteins and methodsof use”);

[0157] WO 99/19470 (“GFP-annexin fusion proteins”)

[0158] WO 98/14605 (“Remilla luciferase and green fluorescent proteinfusion genes”); and

[0159] EP 949269 (“Biosensor protein”).

[0160] V. Applications

[0161] With the provision of redox-sensitive GFP indicators, methods ofmonitoring redox changes in real time in a targeted environment, forinstance in the mitochondria (or other subcellular compartment) ofsingle living cells, are enabled.

[0162] Redox-sensitive GFPs mutant are useful in a wide variety ofapplications, including the monitoring of redox status of individualcells or subcellular compartments. Certain of the provided rosGFPs arealso useful for monitoring pH concurrently with redox status, asexplained herein.

[0163] Mutant GFPs as described herein are suitable for use as markersfor transformation of mammalian cells. Often, a gene of therapeuticinterest does not produce an easily distinguishable phenotype in cellsexpressing that gene. Thus, such a therapeutic gene can be inserted intoa vector that contains a marker gene. The therapeutic gene and themarker gene are placed in the vector under the control of a cellular orviral promoter, and introduced into mammalian cells of interest;subsequently, the transfected cells (the cells containing the vector)are selected according to the phenotype determined by the marker gene.The use of GFP for selection obviates the need to grow the mammaliancells of interest in the presence of drugs in order to select for thetransfected cells. In addition, due to the redox-sensitive nature of theprovided GFP mutants, the redox status of the transformed cells (orrespective subcellular compartment(s)) can be directly measured. Cellstransformed with a nucleic acid comprising a rosGFP can be sorted byFACS.

[0164] For the study of protein localization, fusion of a rosGFP mutantand a sequence encoding a cellular protein (or encoding a fragment,sub-domain, or domain of a protein), and subsequent expression of thefusion construct, results in a fluorescent fusion protein that islocalized at the normal intracellular location of the protein encoded bythe nucleic acid sequence of interest. Identifying the intracellularlocation of the mutant GFP thus identifies the intracellular location ofthe protein of interest. The use of such fusion proteins yieldsinformation on the normal cellular role of the protein encoded by thegene of interest, and provides direct measurement of the redox status(and in some embodiments, pH) of the intracellular environment to whichit was targeted. Such an application using wtGFP is described in moredetail, for example, in an article by Olson et al. (J. Cell Biol.,130:639-650,1995).

[0165] Also provided are rosGFPs that are sensitive to pH, including forinstance rosGFP2, 4, and 6. Dual sensor GFPs such as these can be usedin any situation in which it is beneficial to monitor or measure both pHand redox status in an environment.

[0166] VI. Kits

[0167] Kits are provided that contain at least one rosGFP protein, or anucleic acid molecule (ag., a vector) that encodes such a protein, orboth, in one or more contains. The provided kits may also includewritten instructions. The instructions can provide calibration curves orcharts to compare with the determined (e.g., experimentally measured)values. Included are kits that can be used for diagnosis or prognosis ofa disease or other condition associated with a change in the redoxstatus of cell or sub-cellular compartment.

[0168] The invention is illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Selection and Design of Redox-Sensitive GFPs

[0169] In order to create redox-sensitive GFPs, two cysteine residueswere introduced into GFP that would be within disulfide bonding distanceof each other, near the chromophore, and on the surface of the protein.The cysteine residues bring about fluorescence changes based uponwhether they are reduced or oxidized. In addition, by being on thesurface of the protein, the cysteines are solvent accessible, whichenables them to reversibly respond to redox changes in the surroundingenvironment.

[0170] After close examination of the crystal structure of the S65Tvariant of GFP, two borderline suitable sites for the introduction of apair of cysteines were chosen. The first site chosen was positions 147and 204, while the second site chosen was positions 149 and 202. Allfour amino acid side-chains at these positions pointed away from theprotein's interior. The distance between the C_(α)-C_(α)andC_(β)-C_(β positions for site one were) 4.6 Å each, and for site two thedistance between these positions were 4.8 and 5.9 Å, respectively. Thesedistances did not agree with previous reports on ideal geometry for theintroduction of disulfide bridges in proteins. In addition, neither ofthese site pairs were chosen by a disulfide bridge modeling program(Sowdhamini et al., Protein Eng. 3:95- 103, 1989). There were, however,some indications that one or both of these pairs of cysteines might beable to form a disulfide bond. Such evidence came from the irregular“bulging” nature of the β-strand encompassing positions 147 and 149,which has previously been shown to move in response to substitution atposition 148 (Wachter et al., Structure 6:1267-1277, 1998). Flexibilityhas been suggested to help ensure that a protein can both adjust to theperturbation due to replacements with cysteine residues as well as toallow the disulfide bridge to assume near-optimal geometry (Matsumura etal., Proc. Natl. Acad. Sci. USA 86:6562-6566, 1989). The 147/204 and149/202 sites best fit the criteria.

Example 2 Construction and Expression of Redox Sensitive GFPs

[0171] Wild-type GFP contains two cysteine residues at positions 48 and70. To avoid possible thiol/disulfide interchange reactions with thenewly engineered cysteines, cysteine 48 and cysteine 70 were replacedwith serine and alanine, respectively. Although the substitution C48Sdid not alter the properties of GFP, C70A appeared to be deleterious toobtaining soluble, fluorescent protein. Since position 70 is locatedwithin the interior of GFP and is in close proximity to the chromophore,it was deemed a mutation-sensitive position and was therefore left as acysteine.

[0172] The C48S mutation was introduced into a histidine-tagged versionof the S65T variant of GFP in the plasmid pRSET_(B). This constructserved as the template for introduction of the cysteines at sites one(S147C/Q204C) and two (N149C/S202C). All mutations were introduced viathe QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla,Calif.), following the manufacturer's protocol. Mutations were verifiedby DNA sequencing of the entire GFP coding sequence.

[0173] Mutant protein was recombinantly expressed in Escherichia coli,strain JM109(DE3). Transformed bacteria were grown in four liters ofS-LBH media at 37° C., stirred at 450 rpm, with 5 liters per minute airflow, and in the presence of 0.27 mM ampicillin. After the culturereached a density of approximately OD₅₉₅ equal to 0.8, then proteinexpression was induced by addition of isopropyl-β-Dthiogalactopyranoside (IPTG) to a final concentration of 1 mM. At thesame time the temperature of the culture was reduced to 16° C. and theculture allowed to grow for an addition 16 hours. Cells were thenharvested by centrifugation at 4° C. in a Beckman KA-9.1000 rotor at11,800×g for 10 minutes.

[0174] The bacterial cell pellet was resuspended in 100 mL of 50 mMHEPES (pH 7.9), 300 mM NaCl, 10% glycerol, and 0.1 mMphenylmethylsulfonyl fluoride (PMSF). The resuspended cells weresonicated for a total of five minutes, and the lysate clarified twice bycentrifugation at 35,000×g in a Beckman JA-20 rotor at 4° C. for 20minutes.

[0175] The resultant supernatant was loaded onto a pre-equilibratednickel-nitrilotriacetic acid (Ni-NTA) metal-affinity column (Qiagen,Hilden, Germany). The equilibration and subsequent washing of the columnwas performed with washing buffer (50 mM HEPES (pH 7.9) and 300 mMNaCl). Proteins were eluted from the column by a step gradient of (1)washing buffer plus 20 mM imidazole to remove mostly unwanted proteins,and then (2) washing buffer plus 100 mM imidazole to elute the mutantGFP. To remove the amino-terminals histidine tag and as a filerpurification step the eluted protein was incubated with {fraction(1/50)} w/w γ-chymotrypsin at 22° C. for 22 hours. The proteinpreparation was finally buffer exchanged on a Sephadex® G-25 column.Characteristic yields of mutant GFP protein were in the range of 15 to100 milligrams and with a purity greater than 95%.

Example 3 Disulfide-Bond Formation and Redox Sensitivity of rosGFPs

[0176] Samples of rosGFP2 and GFP S65T (control) were treated with 1 mMDTT or 1 μM CuCl₂ and incubated at room temperature for 3-4 hours, then2 mM N-ethyl maleimide was added to prevent disulfide exchangereactions. Molecular weights were determined by comparison to BenchMark™protein ladder (InVitrogen, Carlsbad, Calif.). The gel was visualizedwith Coomassie blue stain.

[0177] Results

[0178] To verify that the introduced cysteines formed disulfide bondsand to show that the disulfide bonds were intramolecular, SDS-PAGE wasrun on redox-sensitive GFP #2 (rosGFP2), harboring the mutationsC48S/S65T/S147C/Q204C, and on a control GFP (C48S/S65I) under reducingand non-reducing conditions. If an intramolecular disulfide bond forms,the resulting polypeptide would be expected to migrate further based onits slightly more compact structure in the denatured form. However, ifan intermolecular disulfide bridge forms, then a large molecular weightband (˜60 kDa) corresponding to a disulfide-linked dimer, would beexpected.

[0179] The results (FIG. 4) indicate that the introduced cysteines formintramolecular disulfide cross-links, but are unable to producedisulfide-linked dimers. This can be seen by comparing lanes 11 and 12with lanes 14-16 or lanes 21 and 22 with lanes 17-19 (FIG. 4).

Example 4 Determination of Redox Potentials

[0180] Apparent redox potential values for the rosGFPs were found byexploiting the fact that the fluorescence of the rosGFP chromophores isstrongly dependent upon the redox state of the introduced cysteines.Therefore the redox equilibrium of the rosGFPs with dithiothreitol(DTT_(red)) and oxidized dithiothreitol (DTT_(ox)) was analyzed. Theequilibrium for the oxidation of reduced rosGFP by DTT_(red) and itsequilibrium constant (K_(eq)) are given by equations 1 and 2.

rosGFP_(red)+DTT_(ox)⇄rosGFP_(ox)+DTT_(red)  (1)

K_(eq)[rosGFP_(ox)][DTT_(red)]/[rosGFP_(red)][DTT_(ox)]  (2)

[0181] When rosGFPs were incubated in the presence of varyingconcentrations of DTT_(red) and DTT_(ox) (total DTT_(red)+DTT_(ox=)1mM), the fractional amount of reduced rosGFP at equilibrium (R) could bemeasured over the whole range from the oxidized to the reduced proteinusing the chromophore fluorescence (FIG. 20). Based on the SDS-PAGEresult hat indicated rosGFPs only form intramolecular disulfide bonds, Rcan be related to K_(eq) by equation 3 (Hawkins et al., Biochem J 1991275(2):341-348).

R=([DTT_(red)]/[DTT_(ox)]/(K_(eq)+[DTT_(red)]/[DTT_(ox)])  (3)

[0182] For experimental determination of the equilibrium constants ofthe GFP:dithiothreitol system, the equilibrium concentrations ofDTT_(red) and DTT_(ox) were calculated according to equations 4-6, where[DTT_(red o)] and [DTT_(ox o)] are the initial concentrations ofDTT_(red) and DTT_(ox). respectively, R is the fractional amount ofreduced rosGFP at equilibrium, [rosGFP_(o)] is the initial concentrationof oxidized rosGFP, F is a fluorescence intensity ratio of band Bexcitation (490 nm) versus the isosbestic point (425 nm), and F_(ox),and F_(red) are the 490:425 nm ratios of the completely oxidized andreduced protein, respectively. By plotting R against the

[DTT_(red)][DTT_(red o)]−R[rosGFP_(o)]  (4)

[DTT_(ox)]=[DTT_(ox o)]+R[rosGFP_(o)]  (5)

R=(F−F _(ox))/(F _(red) −F _(ox))  (6)

[0183] [DTT_(red)]/[DTT_(ox)] ratio and fitting the data to a titrationcurve according to equation 3 (FIG. 21), the K_(eq) for therosGFP1:dithiothreitol system was found to be 2.05×10⁻². The redoxpotential of rosGFP2 at pH 7 and 30° C. (E_(o)″_(rosGFP2)) was thencalculated from the Nernst equation (7), where E_(o)″ is thebiochemist's standard potential of the DTT/DTT_(ox) couple(E_(oDTT=−)0.323 V, at pH 7 and 30° C.; Szajewski and Whitesides 1980),R is the gas constant (8.315 J K⁻¹ mol⁻¹), T is the absolute temperature(303.15 K), n is the number of transferred electrons (2), and F is theFaraday constant (9.649×10⁴ C mol⁻¹) and found to be −0.272 V.

E _(o)′_(rosGFP2) =E _(o)′_(DTT)−(RT/nF)×ln K _(eq)  (7)

[0184] Redox potentials involving the liberation of H⁺ ions areintrinsically based on pH. The pH-dependence on the redox potential ismore apparent when examining the two half-reactions involving rosGFP andDTT (equation 8 and 9).

rosGFP_(ox)+2H⁺+2e⁻⇄rosGFP_(red)   (8)

DTT_(red)⇄DTT_(ox)+2H₊2e⁻  (9)

[0185] At equilibrium the concentrations of rosGFP_(ox) and rosGFP_(red)are equal and thus the K_(eq) is equal to the [H+]². The standard redoxpotential of rosGFP2 (E_(o rosGFP2)) at pH 0 could then be determinedfrom equation 10.

E _(o rosGFP2) =E _(o)″_(rosGFP2)−(RT/nF)×ln K _(eq)  (10)

[0186] E_(o rosGFP2) was calculated to be 0.149 V. Equation 11simplifies the expression for the pH-dependence of redox potentialsinvolving two protons.

E _(o) ^(pH) =E _(o)″−60.2 mV×(pH−7)  (11)

[0187] The pH-dependence on the redox potential therefore changes 60.2mV with each pH unit. Experimentally E_(o rosGFP2) varied 65.5 mV per pHunit from pH 6 to 8 (correlation coefficient: 0.9999). Therefore,although a linear correlation between pH and E_(o rosGF2) is observed,the pH-dependence of the rosGFP2 standard potential does not directlycorrespond to this model. The deviation from this model for thepH-dependence of E_(o) may be due in part to the contributions ofcharged residues near the introduced disulfide of rosGFP2 as well aspotentially different pK_(a) values for the cysteine residues(Wunderlich and Glockshuber, Protein Sci 2(5):717-726, 1993; Wunderlichand Glockshuber, J Blot Chem 268(33):24547-24550, 1993)

Example 5 Spectral Analysis of rosGFPs

[0188] Spectroscopy and pH Titrations

[0189] Absorbance measurements were performed on a Shimadzu 2101spectrophotometer. The molar extinction coefficient of GFP S65T(λ_(280 nm)=19,890 N⁻¹cm⁻¹) was calculated from its amino acid sequenceas previously described (Gill and von Hippel, Anal. Biochem.182:319-326, 1989), and used to determine protein concentrations of therosGFPs. pH titrations were performed using approximately 200 μg mL⁻¹mutant GFP in 75 mM buffer, 140 mM NaCl and either 1 μM CuCl₂ or 5 mMDTT. According to the desired pH, an appropriate buffer was chosen fromMES, HEPES, or CHES and the final pH was adjusted by addition of HCl orNaOH. The absorbance was then scanned between 250 and 550 nm and theoptical density of the long-wave band B was plotted as a function of pHand fitted to a titration curve to obtain pK_(a) values.

[0190] Fluorescence excitation spectra at various pHs were attained on aHitachi F4500 fluorescence spectrophotometer or a Perkin Elmer LS 55luminescence spectrometer at protein concentrations of approximately 100μg mL⁻¹ in the same buffers used for absorbance measurements. Apparentchromophore pK_(a) values were determined by plotting the emissionintensity when excited at band B as a function of pH and fitting thedata to a titration curve (Kaleidagraph™). In all cases, the pK_(a)values determined by absorbance and fluorescence differed by no morethan +/−0.05 of a pH unit. All plots and curve fits were made withKaleidagraph™ (Abelbeck Software).

[0191] Redox Titrations

[0192] Fluorescence measurements were performed at 30° C. using athemostated cuvette holder. Samples consisted of 1 μM GFP in 75 mM HEPES(pH 7.0), 140 mM NaCl, 1 mM EDTA, and 1 mM total DTT (mixture ofoxidized and reduced forms). To exclude air oxidation, the solutionswere degassed and subsequently flushed with nitrogen. In generalequilibration was reached within one hour at pH 7.0. Equilibration ofrosGFPs was ensured by incubating the samples at 30° C. for four hours.The reaction appeared to be at equilibrium, since the ratio of oxidizedand reduced protein, as determined by fluorescence, did not changebetween 4 and 16 hour incubation times.

[0193] Spectral Characteristics

[0194] To test whether the introduced cysteines affect the spectralproperties of GFP, absorbance and fluorescence scans were collected atvarying redox potentials. To establish various redox potentials, GFP wasequilibrated with varying concentrations of dithiothreitol (DTT) andoxidized DTT (DTT_(ox)) FIG. 5 shows the absorbance and fluorescenceexcitation spectra of rosGFP2 as a function of redox potential. With adisulfide bond formed under oxidizing conditions, the 400 nm peak (bandA) was maximized whereas the 490 nm peak (band B) was minimized.Conversely, in the absence of a disulfide cross-link achieved withreducing conditions, band B was at a maximum, while band A was at aminimum. The two peaks were separated by a clean isosbestic point at 425nm, indicative of two species interconverting. The redox potential ofrosGFP2 was then determined from the equilibrium constant obtained byplotting the fraction of reduced protein versus the ratio of DTT_(red)to DTT_(ox) (see Example 4) and discovered to be −0.272 V.

[0195] Introducing a pair of cysteines at the alternative site alsoyielded a mutant protein whose fluorescent properties varied in responseto redox potential (FIG. 6A). The rosGFP4 variant, having the mutationsC48S/S65T/N149C/S202C, displayed increased band B fluorescence overrosGFP2, however band A fluorescence intensity was almost non-existentThe fluorescence isosbestic point of rosGFP4 was shifted toapproximately 400 nm (FIG. 6B).

[0196] Combining the two pairs of cysteine substitutions resulted in avariant (C48S/S65T/S147C/N149C/S202C/Q204C, rosGFP6), whose propertiesappeared to be an average of both rosGFP2 and rosGFP4. FIG. 7 shows thefluorescence excitation spectra of rosGFP6. In this variant the dynamicrange of the two excitation peaks as well as the isosbestic point lie inthe middle of the values observed for either rosGFP2 or rosGFP4.

[0197] The overall dynamic range of the excitation ratio (δ), determinedby dividing the maximum and minimum possible excitation peak ratios, wasfound to be 5.4 for rosGFP6. Table 5 summarizes the spectroscopic andbiochemical parameters of the rosGFP variants. TABLE 5 Spetroscopic andBiochemical Properties of rosGFPs. Name Mutations¹ K_(eq) ² E_(o) ¹(V)³δ⁴ rosGFP2 S65T/S147C/Q204C 0.0205 −0.272 5.8 rosGFP1 S147C/Q204C 0.0702−0.288 6.1 rosGFP4 S65T/N149C/S202C 0.0561 −0.286 2.6 rosGFP3N149C/S202C 0.1505 −0.299 4.3 rosGFP6 S65T/S147C/N149C/S202C/Q204C0.0385 −0.280 5.4 rosGFP5 S147C/N149C/S202C/Q204C 0.1341 −0.296 7.8

[0198] pH-Sensitivity of rosGFPs

[0199] Previous work has shown that, while the fluorescence of wild-typeGFP is unaffected throughout the biologically relevant pH range from 6to 8 (Ward and Bokman, Photochem. Photobiol. 35:803-808, 1982), GFPvariants harboring the S65T mutation often exhibit dramatic fluorescencechanges over this pH range (Kneen et al., Biophys. J 74:1591-1599,1998;Wachter et al., Structure 6:1267-1277, 1998; Elsliger et al.,Biochemistry 38:5296-5301, 1999). To determine if the rosGFPs were alsopH-sensitive, their absorbance and fluorescence were scanned over a widerange of pHs. FIG. 8 shows both absorbance and fluorescence excitationspectra of oxidized rosGFP2 versus pH. The spectra show that, as the pHis increased from 5.2 up to 9.0, the fluorescence intensity of bothexcitation peaks increases. Plotting the intensity at 490 nm as afunction of pH and fitting this to a titration curve with a singleionization constant gave a pK_(a) value of 6.0 (FIG. 9). Titration ofrosGFP2 in the reduced state shifted the pK_(a) to 5.6 (FIG. 10).

[0200] Initially it appeared that the pH-sensitivity of the rosGFPsmight pose a problem for using them as tools to determine in vivo redoxpotentials by introducing pH artifacts. Therefore, to produce apH-insensitive rosGFP, threonine 65 was reverted back to serine, whichis the amino acid found at this position in wild-type GFP.

[0201] Not only did the T65S reversion completely eliminatepH-sensitivity over the range of 6 to 8, but it greatly altered thespectral properties of the rosGFPs. FIGS. 11, 12, and 13 show thefluorescence excitation spectra of rosGFP1 (C48S/S147C/Q204C), rosGFP3(C48S/N149C/S202C), and rosGFP5 (C48S/S147C/N149C/S202C/Q204C) atvarying redox potentials. The most striking difference between theserosGFPs and threonine 65 containing rosGFPs is the favoring of band Aover band B fluorescence. There is also a tendency toward more evenexcitation of both bands, which is especially evident in the rosGFP3 androsGFP5 variants. Unexpectedly, the T65S reversion led to a 13-16 mVmore reducing redox potential (see Table 5).

[0202] Subsequent to construction of the variants containing the T65Sreversion, it was determined that, by using a fluorescence excitationratio, any effects induced by pH changes were virtually eliminated. FIG.14 illustrates how the ratio of excitation wavelengths remains aconstant over a large pH range in both the fully reduced or fullyoxidized protein. In other words, the fluorescence increase due toalkalization or fluorescence decrease upon acidification seen in theexcitation spectra of rosGFP2 affects the entire spectrum in the samemanner, and hence the ratio of intensities is unaffected by pH changesin the range of at least pH 6 to 8.5. Therefore, a ratio of theexcitation wavelengths cancels out variations due to pH fluctuations andallows for redox status determination in the absence of pH artifacts.

[0203] Since redox reactions involving the liberation of H⁺ ions areintrinsically based on pH (see Example 4), and consequently the pH ofthe cellular compartment of study must be determined, the ability toseparate out the pH and redox sensing capabilities of rosGFP2 wasinvestigated. Excitation of band A was found to result in pH-dependantdual-emission (FIG. 15). In other words, at low pH, blue emissionprevails and at high pH, green emission dominates, with pK_(a)s of 5.6(reduced) to 6.0 (oxidized) dependant upon the redox state of rosGFP2.This phenomenon is a useful means of monitoring pH changes in vivo. Byutilizing a ratio of emission wavelengths, pH variations due to theredox state of the probe are minimized (FIG. 16). Therefore, it ispossible to experimentally separate out the pH and redox contributionsto the fluorescent signal, and thus simultaneous monitoring of redox andpH is possible with the same probe.

Example 6 Structural Basis for Dual-Excitation

[0204] The crystal structure of rosGFP2 was solved in order to betterunderstand how disulfide bond formation leads to dual excitation.Crystals belonging to the space group P2₁2₁2 were grown of both theoxidized and reduced forms of rosGFP2 and the crystal structures weresolved by molecular replacement to 1.9 Å and 2.0 Å, respectively. Theprotein crystallized as a dimer, with three molecules in the asymmetricunit. One molecule is related to its dimer mate by a crystallographictwo-fold axis of symmetry, and the other dimer is related to itself by anon-crystallographic two-fold axis of symmetry.

[0205] Since the resolution limit of the data collected was fairly high,refinement was performed without imposing any non-crystallographicsymmetry constraints, and thus each of the three molecules in theasymmetric unit were independently refined. Analysis of the differentmolecules in the asymmetric unit after refinement revealed only verysubtle differences, all of which were within experimental error(approximately 0.2 Å) for the atomic positions at his resolution.Interestingly, the surface facing a solvent channel of one of themolecules comprising the non-crystallographic symmetry dimer is verypoorly ordered This disorder is most certainly reflected in many of therefinement statistics presented in Table 6. TABLE 6 Data Collection andRefinement Statistics for Oxidized and Reduced rosGFP2. Data CollectionCrystal Oxidized Reduced Total observations 203,865 194,884 Uniquereflections 56,854 38,346 Cell dimensions 186.84, 67.61, 56.08 185.63,67.86, 56.38 (a, b, c; Å) Resolution (Å) 29.7-1.90 28.7-2.00 Highestresolution shell 1.95-1.90 2.10-2.00 (Å) Completeness¹ (%) 99.8 (100)78.2 (74.3) Multiplicity¹ 3.6 (3.6) 5.1 (5.5) Average I/σ¹ 7.2 (2.3) 8.7(2.2) R_(merge) ^(1,2) 0.058 (0.247) 0.053 (0.304) Refinement SpacegroupP2₁2₁2 P2₁2₁2 Number of molecules³ 3 3 Number of protein 5,220 5,216atoms³ Number of solvent 174 132 atoms³ R_(factor) ⁴ 0.229 0.223 AverageB-factors (Å²) 46.6 49.7 Protein atoms 46.7 49.7 Solvent 44.2 47.4 rmsdeviations Bond lengths (Å) 0.020 0.014 Bond angles (°) 3.111 2.436B-factor correlations (Å²) 7.019 5.779

[0206] The dimer interface is essentially the same as seen for wild-typeGFP and the yellow variant of GFP (Yang et al., Nature Biotech. 14:1246-1251, 1996; Wachter et al., Structure 6:1267-1277, 1998). One moleculeof the dimer is tilted approximately 70 degrees with respect to theother molecule based on an imaginary axis drawn from one end of the GFPbarrel structure through the center and out the other end. The dimerinterface is comprised of a small hydrophobic patch consisting ofalanine 206, leucine 221 and phenylalanine 223, a hydrogen bondinteraction between tyrosine 39 and aspartame 149, as well as a numberof hydrophilic contacts involving several bound water molecules. Atpresent it is not known whether the dimer is solution relevant or simplyan artifact created by the crystallization conditions (Palm andWlodawer, Nat. Struct. Biol. 4:361-365, 1997). At any rate, knowledge ofthe dimer interface residues permits future mutational studies aimed atdisrupting dimers without affecting protein stability and folding.

[0207] As expected, the introduced cysteines are positioned toward theoutside of the protein and are in excellent arrangement to form adisulfide bond under oxidizing conditions. They reside along the edge ofthe dimer interface and adjacent to bulk solvent. The individualdisulfides are separated by 14 ™ from the opposing dimer pair ofcysteines and therefore, as shown by gel electrophoresis, are unable toform intermolecular disulfide bonds and are unlikely to aid in dimerformation. The disulfide bond present in the oxidized structure does nothave ideal geometry, with an average C_(α)-C_(α) distance of 4.0 Å,C_(β)-C_(β) distance of 4.2 Å, S-S distance of 2.0 Å, C_(β)-S angle of112°, and C_(β)-S-S angle of 106°. These parameters diverge from thosevalues seen in other structures of disulfide bond-containing proteins(Sowdhamini et al. 1989; Matsumura et al. 1989), and may also accountfor or contribute to the observed spectral perturbations.

Example 7 Mammalian Cell Expression and Fluorescence Microscopy

[0208] The mutations C48S/T65S/S147C/Q204C were introduced into themammalian expression plasmid pEGFP-N1 (CLONTECH, Palo Alto, Calif.).This plasmid has the “folding mutation” F64L, which was found not toalter the spectral or redox properties of the rosGFPs. HeLa cellstransiently transfected with this plasmid using Fugene(Boehringer-Mannheim, Germany) were imaged one day post-transfection ona motorized Zeiss Axioscope 2 microscope. The temperature of the cellswas maintained at 37° C. using an open perfusion micro-incubator(Harvard Apparatus Inc., Holliston, Mass.). Dual-excitation ratioimaging required 400(10) and D480/30x excitation filters, a 505DCXRdichroic mirror, and a D535/40m emission filter (Edmund ScientificCompany, Omega Optical and Chroma Technologies, Battleboro, Vt.)alternated by a fast filter changer. Images were collected with aPentaMax cooled CCD camera (Princeton Instruments). Data was collectedand processed using the program Openlab (Improvision, Lexington, Mass.).

[0209] In vivo Redox Status

[0210] To determine if redox-sensitive GFPs work as indicators of redoxstatus within mammalian cells, rosGFP1 was expressed in the mitochondriaof cultured HeLa cells. FIG. 17 shows the reticular localization patternof rosGFP1 expressed in the mitochondrial matrix via fusion at the DNAlevel to the mitochondrial targeting sequence of the E₁α subunit ofpyruvate dehydrogenase. Upon addition of an oxidizing agent (H₂O₂) thefluorescence excitation ratio (400 nm/490 nm) increased, as expected foran oxidation event (FIG. 18). Conversely, the addition of a reducingagent (DTT) decreased the elevated ratio to a level below the initialratio.

[0211] From this experiment several things can be concluded. First,rosGFP1 is able to reversibly respond to induced redox changes withinliving cells. As seen in FIG. 18, large fluorescence changes accompanythe addition of H₂O₂ or DTT. Second, the results demonstrate thatinitially rosGFP1 is not fully reduced or oxidized within mitochondria.Therefore rosGFP1 is able to detect the intrinsic redox potential insidemitochondria, presumably by interacting with endogenous oxidizing andreducing agents. Moreover, the redox potential of rosGFP1 is close tothat of mitochondria and hence rosGFP1 should make an excellent probefor studying redox changes in mitochondria. Finally, given the initialfluorescence excitation ratio, the percentage of reduced to oxidizedrosGFP1 could be estimated and thus the in vivo redox potential ofmitochondria could be calculated.

[0212] Since redox potentials involving the liberation of H⁺ ions areintrinsically based on pH (see Example 4), the GFP S65T/H148D variant(Wachter el at., Structure 6:1267-1277, 1998), with a pK_(a) of 7.8, wasused to examine the pH within the mitochondrion. The inherent pH sensingabilities of rosGFP2 was not utilized in this instance, due to the highpH nature of mitochondria and the high levels of autofluorescence. Thefluorescence excitation ratio of GFP S65T/H148D changed as function ofpH inside the mitochondrial matrix. The addition of oligomycin,carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), orhydrochloric acid resulted in an acidification of the mitochondrialmatrix. On the other hand, addition of NH₄Cl caused the matrix pH toincrease. From these types of additions, the resting pH of themitochondrial matrix of HeLa cells was estimated to be 7.65±0.15 (n=110cells from seven experiments). Due to a slow acidification processduring the experimental setup and data collection process, this valuemay be an underestimate of the true mitochondrial matrix pH. Therefore,the pH value of 7.98±0.07 estimated by Llopis et al. for themitochondrial matrix of HeLa cells was used in subsequent calculations(Llopis et al., Proc. Natl Acad Sci. USA 95:6803-6808, 1998).

[0213] Based on an average of 23 cells from seven independentexperiments, and accounting for the 60.2 mV per pH unit change in redoxpotential for a reaction involving two protons (see Example 4), theredox potential in normal resting HeLa cell mitochondria was determinedto be substantially more reducing than −0.3 V.

[0214] To investigate the redox potential of the cytosol, rosGFP2 wasexpressed in the cytosol of HeLa cells. The starting fluorescenceamplitude ratio of 400/480 nm excitation was low, as expected for thereducing environment of the cytosol of healthy cells. Again, there was amarked increase and decrease in the ratio upon addition of H₂O₂ and DTT,respectively. However the ratio only recovered to the starting value,indicating that rosGFP2 was fully reduced in the cytosol. Hence theredox potential of the cytosol was estimated to be more reducing than−0.30 V, assuming a pH of 7.34 (Llopis el al., Proc. Natl. Acad Sci. USA95:6803-6808, 1998).

Example 8 Factors that Influence the Redox State of rosGFPs In vivo

[0215] It is generally thought that the major redox buffer in cells isglutathione and that the ratio of reduced glutathione (GSH) toglutathione disulfide (GSSG) is the crucial parameter for determiningredox status (Meister, Methods Enzymol. 251:3-7, 1995; Deneke, Curr.Top. Cell. Regul. 36:151-180, 2000). On average, the ratio of GSH toGSSG is greater than 100:1 for whole cell determinations on a widevariety of tissue types and total glutathione levels are believed to bearound 1-10 mM (Kosower and Kosower, Int. Rev. Cytol. 54:109-160, 1976;Voet and Voet, Biochemisty, 2nd ed. John Wiley & Sons, Inc., 1995, NewYork, N.Y.; Meister, Methods Enzymol. 251:3-7, 1995). To investigatewhether glutathione may contribute to the redox state of rosGFPs invivo, fluorescence experiments were conducted in vitro.

[0216] Incubation of reduced rosGFP1 with GSSG resulted in completeoxidation of rosGFP1 in the absence of air oxidation. From thisexperiment it was concluded that rosGFPs expressed in vivo can beoxidized by GSSG. However, since rosGFPs have a lower affinity forelectrons than does glutathione, GSH is unable to reduce oxidizedrosGFPs.

[0217] Several reports agree that the ratio of GSH to GSSG inmitochondria is between 6:1 to 33:1, with total glutathioneapproximately 1-2 mM (LêQuôc and LêQuôc, Arch. Biochem. Biophys.273:466-478, 1989; Bindoli et al., Arch Biochem. Biophys. 342:22-28,1997; Lenton et al., Anal. Biochem. 274:125-130, 1999). Substitutingthese values into the Nernst equation (assuming a mitochondrial pH of7.98, 37° C., and a standard redox potential of −0.205 V (Szajewski andWhitesides, J. Am. Chem. Soc. 102:2011-2026, 1980) for the GSH/GSSGcouple) results in a mitochondrial redox potential in the range of−0.210 to −0.230 V. Since the redox potential of mitochondria isestimated to be much more reducing than −0.3 V, glutathione alone doesnot appear to account for such a reducing potential. Therefore, otherredox active agents were investigated.

[0218] The NAD⁺/NADH (nicotinamide adenine dinucleotide) pair wasconsidered, because it is present at high concentrations in mitochondriaand has a very reducing redox potential. The ratio of NAD⁺to NADH in thepresence or absence of glucose has been estimated to be anywhere from1:6 to 10:1 in mitochondria (LêQuôc and LêQuôc, Arch. Biochem. Biophys.273:466-478, 1989; Ramirez et al., Biochim. Biophys. Acta 1273:263-267,1996; Robinson, Methods Enzymol. 264:454-464, 1996). These ratiostranslate into redox potentials in the range of −0.328 to −0.382 V at pH7.98 and 37° C. using −0.320 V as the standard potential of theAND⁺/NADH couple at pH 7. As a result, this redox couple appeared to bean ideal candidate for maintaining the very reducing environment of themitochondrion. However, redox reactions involving nicotinamides carryout concerted two-electron transfers, whereas thiols undergo twosequential one-electron transfers. As expected, NADH was experimentallyunable to directly reduce rosGFP1 in vitro.

[0219] There exists a family of pyridine nucleotide-disulfideoxidoreductases, comprising lipoamide dehydrogenases (LDH), glutathionereductases, thioredoxin reductases, trypanothione reductase, andalkylhydroperoxide reductase. These enzymes all perform homologousreactions ultimately involving the transfer of NADH or NADPH(nicotinamide adenine dinucleotide phosphate) reducing equivalents tothiols (Carothers et al., Arch. Biochem. Biophys. 268:409-425, 1989). Ingeneral terms, the reducing equivalents are transferred from NADH orNADPH through a concerted two-electron transfer reaction to a bound FAD(flavin adenine dinucleotide) cofactor. FAD then reduces a nearbydisulfide bridge through two sequential one-electron transfer reactions.In some cases, the electrons are transferred by disulfide exchange toother nearby disulfides (Ellis and Poole, Biochemistry 36:13349-13356,1997; Calzi and Poole, Biochemisty 36:13357-13364, 1997). Finally, thereduced pair of thiols participates in the reduction of a substrate suchas lipoamide, glutathione, thioredoxin, or one of various peroxides. Thekey to how these enzymes carry out their electron transfer reactions islinked to the chemistry of the FAD cofactor, which is able to undergotwo sequential one-electron transfers or a simultaneous two-electrontransfer.

[0220] To determine if rosGFPs may be in equilibrium with agents otherthan just glutathione an in vitro system analogous to a portion of thepyruvate dehydrogenase complex was set up. The system consisted of LDH,lipoate, NADH, and oxidized rosGFP1. The rationale behind thisexperimental setup was to monitor the reduction of rosGFP1 by NADH,through the LDH enzyme ad free lipoic acid. FIG. 19 shows that, with allfour components of the system present, nearly 75% of rosGFP1 is reduced,however removal of LDH, lipoate, or NADH results in less than 5%reduction of rosGFP1. Only partial reduction was expected since theredox potential of rosGFP1 (−0.288 V) and the lipoic acid/dihydrolipoicacid couple (−0.29 V; Szajewski and Whitesides, J. Am. Chem. Soc.102:2011-2026, 1980) are very similar. Air oxidized and DTT reducedrosGFP1 were used as standards for obtaining the zero and 100% values.The results of this experiment indicate that it may be possible for NADHreducing equivalents, through various enzymes such as pyruvatedehydrogenase, to be ultimately imposed on rosGFPs in vivo.

[0221] This disclosure provides redox-sensitive green fluorescentproteins (rosGFPs), nucleic acids encoding these proteins, and cellstransformed with a nucleic acid encoding a rosGFP. The disclosure fakerprovides methods of using these molecules to analyze the redox statusof, for instance, a cell or subcellular compartment. It will be apparentthat the precise details of the methods described may be varied ormodified without departing from the spirit of the described subjectmatter. We claim all such modifications and variations that fill withinthe scope and spirit of the claims below.

1 13 1 238 PRT Aequorea victoria 1 Met Ser Lys Gly Glu Glu Leu Phe ThrGly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn GlyHis Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr GlyLys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val ProTrp Pro Thr Leu Val Thr Thr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe SerArg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser AlaMet Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Tyr Lys Asp AspGly Asn Tyr Lys Ser Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp ThrLeu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu AspGly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140 Tyr Asn Ser HisAsn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile LysVal Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 GlnLeu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val 210215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230235 2 717 DNA Artificial Sequence Variant derived from A. victoria 2 atgagt aaa gga gaa gaa ctt ttc act gga gtt gtc cca att ctt gtt 48 Met SerLys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 gaatta gat ggt gat gtt aat ggg cac aaa ttt tct gtc agt gga gag 96 Glu LeuAsp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt gaaggt gat gca aca tac gga aaa ctt acc ctt aaa ttt att tcc 144 Gly Glu GlyAsp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Ser 35 40 45 act act ggaaaa cta cct gtt cca tgg cca aca ctt gtc act act ttc 192 Thr Thr Gly LysLeu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 act tat ggt gttcaa tgc ttt tca aga tac cca gat cat atg aaa cgg 240 Thr Tyr Gly Val GlnCys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 cat gac ttt ttcaag agt gcc atg ccc gaa ggt tat gta cag gaa aga 288 His Asp Phe Phe LysSer Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act ata ttt ttc aaagat gac ggg aac tac aag aca cgt gct gaa gtc 336 Thr Ile Phe Phe Lys AspAsp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggt gatacc ctt gtt aat aga atc gag tta aaa ggt att 384 Lys Phe Glu Gly Asp ThrLeu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaa gatgga aac att ctt gga cac aaa ttg gaa tac aac 432 Asp Phe Lys Glu Asp GlyAsn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 tat aac tgc cac aatgta tac atc atg gca gac aaa caa aag aat gga 480 Tyr Asn Cys His Asn ValTyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 atc aaa gtt aacttc aaa att aga cac aac att gaa gat gga agc gtt 528 Ile Lys Val Asn PheLys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 caa cta gca gaccat tat caa caa aat act cca att ggc gat ggc cct 576 Gln Leu Ala Asp HisTyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt tta ccagac aac cat tac ctg tcc aca tgc tct gcc ctt tcg 624 Val Leu Leu Pro AspAsn His Tyr Leu Ser Thr Cys Ser Ala Leu Ser 195 200 205 aaa gat ccc aacgaa aag aga gac cac atg gtc ctt ctt gag ttt gta 672 Lys Asp Pro Asn GluLys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 aca gct gct gggatt aca cat ggc atg gat gaa cta tac aaa taa 717 Thr Ala Ala Gly Ile ThrHis Gly Met Asp Glu Leu Tyr Lys 225 230 235 3 238 PRT ArtificialSequence Variant derived from A. victoria 3 Met Ser Lys Gly Glu Glu LeuPhe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp ValAsn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala ThrTyr Gly Lys Leu Thr Leu Lys Phe Ile Ser 35 40 45 Thr Thr Gly Lys Leu ProVal Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Thr Tyr Gly Val Gln CysPhe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 His Asp Phe Phe LysSer Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe LysAsp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu GlyAsp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe LysGlu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr AsnCys His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Cys Ser Ala Leu Ser195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu PheVal 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys225 230 235 4 717 DNA Artificial sequence Variant derived from A.victoria 4 atg agt aaa gga gaa gaa ctt ttc act gga gtt gtc cca att cttgtt 48 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 15 10 15 gaa tta gat ggt gat gtt aat ggg cac aaa ttt tct gtc agt gga gag96 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 2530 ggt gaa ggt gat gca aca tac gga aaa ctt acc ctt aaa ttt att tcc 144Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Ser 35 40 45act act gga aaa cta cct gtt cca tgg cca aca ctt gtc act act ttc 192 ThrThr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 tcttat ggt gtt caa tgc ttt tca aga tac cca gat cat atg aaa cgg 240 Ser TyrGly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 catgac ttt ttc aag agt gcc atg ccc gaa ggt tat gta cag gaa aga 288 His AspPhe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act atattt ttc aaa gat gac ggg aac tac aag aca cgt gct gaa gtc 336 Thr Ile PhePhe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag tttgaa ggt gat acc ctt gtt aat aga atc gag tta aaa ggt att 384 Lys Phe GluGly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gat tttaaa gaa gat gga aac att ctt gga cac aaa ttg gaa tac aac 432 Asp Phe LysGlu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 tat aactgc cac aat gta tac atc atg gca gac aaa caa aag aat gga 480 Tyr Asn CysHis Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 atcaaa gtt aac ttc aaa att aga cac aac att gaa gat gga agc gtt 528 Ile LysVal Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 caacta gca gac cat tat caa caa aat act cca att ggc gat ggc cct 576 Gln LeuAla Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtcctt tta cca gac aac cat tac ctg tcc aca tgc tct gcc ctt tcg 624 Val LeuLeu Pro Asp Asn His Tyr Leu Ser Thr Cys Ser Ala Leu Ser 195 200 205 aaagat ccc aac gaa aag aga gac cac atg gtc ctt ctt gag ttt gta 672 Lys AspPro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 acagct gct ggg att aca cat ggc atg gat gaa cta tac aaa taa 717 Thr Ala AlaGly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 5 238 PRTArtificial sequence Variant derived from A. victoria 5 Met Ser Lys GlyGlu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu AspGly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu GlyAsp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Ser 35 40 45 Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Ser Tyr GlyVal Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 His AspPhe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr IlePhe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 LysPhe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135140 Tyr Asn Cys His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly SerVal 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly AspGly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Cys SerAla Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val LeuLeu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp GluLeu Tyr Lys 225 230 235 6 717 DNA Artificial sequence Variant derivedfrom A. victoria 6 atg agt aaa gga gaa gaa ctt ttc act gga gtt gtc ccaatt ctt gtt 48 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro IleLeu Val 1 5 10 15 gaa tta gat ggt gat gtt aat ggg cac aaa ttt tct gtcagt gga gag 96 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val SerGly Glu 20 25 30 ggt gaa ggt gat gca aca tac gga aaa ctt acc ctt aaa tttatt tcc 144 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe IleSer 35 40 45 act act gga aaa cta cct gtt cca tgg cca aca ctt gtc act actttc 192 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe50 55 60 act tat ggt gtt caa tgc ttt tca aga tac cca gat cat atg aaa cgg240 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 6570 75 80 cat gac ttt ttc aag agt gcc atg ccc gaa ggt tat gta cag gaa aga288 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 8590 95 act ata ttt ttc aaa gat gac ggg aac tac aag aca cgt gct gaa gtc336 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100105 110 aag ttt gaa ggt gat acc ctt gtt aat aga atc gag tta aaa ggt att384 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115120 125 gat ttt aaa gaa gat gga aac att ctt gga cac aaa ttg gaa tac aac432 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130135 140 tat aac tca cac tgt gta tac atc atg gca gac aaa caa aag aat gga480 Tyr Asn Ser His Cys Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145150 155 160 atc aaa gtt aac ttc aaa att aga cac aac att gaa gat gga agcgtt 528 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val165 170 175 caa cta gca gac cat tat caa caa aat act cca att ggc gat ggccct 576 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro180 185 190 gtc ctt tta cca gac aac cat tac ctg tgc aca caa tct gcc ctttcg 624 Val Leu Leu Pro Asp Asn His Tyr Leu Cys Thr Gln Ser Ala Leu Ser195 200 205 aaa gat ccc aac gaa aag aga gac cac atg gtc ctt ctt gag tttgta 672 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val210 215 220 aca gct gct ggg att aca cat ggc atg gat gaa cta tac aaa taa717 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 2357 238 PRT Artificial sequence Variant derived from A. victoria 7 Met SerLys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 GluLeu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 GlyGlu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Ser 35 40 45 ThrThr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 ThrTyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn130 135 140 Tyr Asn Ser His Cys Val Tyr Ile Met Ala Asp Lys Gln Lys AsnGly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu AspGly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro IleGly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Cys ThrGln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His MetVal Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly MetAsp Glu Leu Tyr Lys 225 230 235 8 717 DNA Artificial sequence Variantderived from A. victoria 8 atg agt aaa gga gaa gaa ctt ttc act gga gttgtc cca att ctt gtt 48 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val ValPro Ile Leu Val 1 5 10 15 gaa tta gat ggt gat gtt aat ggg cac aaa ttttct gtc agt gga gag 96 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe SerVal Ser Gly Glu 20 25 30 ggt gaa ggt gat gca aca tac gga aaa ctt acc cttaaa ttt att tcc 144 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu LysPhe Ile Ser 35 40 45 act act gga aaa cta cct gtt cca tgg cca aca ctt gtcact act ttc 192 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val ThrThr Phe 50 55 60 tct tat ggt gtt caa tgc ttt tca aga tac cca gat cat atgaaa cgg 240 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met LysArg 65 70 75 80 cat gac ttt ttc aag agt gcc atg ccc gaa ggt tat gta caggaa aga 288 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln GluArg 85 90 95 act ata ttt ttc aaa gat gac ggg aac tac aag aca cgt gct gaagtc 336 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val100 105 110 aag ttt gaa ggt gat acc ctt gtt aat aga atc gag tta aaa ggtatt 384 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile115 120 125 gat ttt aaa gaa gat gga aac att ctt gga cac aaa ttg gaa tacaac 432 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn130 135 140 tat aac tca cac tgt gta tac atc atg gca gac aaa caa aag aatgga 480 Tyr Asn Ser His Cys Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly145 150 155 160 atc aaa gtt aac ttc aaa att aga cac aac att gaa gat ggaagc gtt 528 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly SerVal 165 170 175 caa cta gca gac cat tat caa caa aat act cca att ggc gatggc cct 576 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp GlyPro 180 185 190 gtc ctt tta cca gac aac cat tac ctg tgc aca caa tct gccctt tcg 624 Val Leu Leu Pro Asp Asn His Tyr Leu Cys Thr Gln Ser Ala LeuSer 195 200 205 aaa gat ccc aac gaa aag aga gac cac atg gtc ctt ctt gagttt gta 672 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu PheVal 210 215 220 aca gct gct ggg att aca cat ggc atg gat gaa cta tac aaataa 717 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230235 9 238 PRT Artificial sequence Variant derived from A. victoria 9 MetSer Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Ser 35 40 45Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 7580 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 9095 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu TyrAsn 130 135 140 Tyr Asn Ser His Cys Val Tyr Ile Met Ala Asp Lys Gln LysAsn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile GluAsp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr ProIle Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu CysThr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp HisMet Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His GlyMet Asp Glu Leu Tyr Lys 225 230 235 10 717 DNA Artificial sequenceVariant derived from A. victoria 10 atg agt aaa gga gaa gaa ctt ttc actgga gtt gtc cca att ctt gtt 48 Met Ser Lys Gly Glu Glu Leu Phe Thr GlyVal Val Pro Ile Leu Val 1 5 10 15 gaa tta gat ggt gat gtt aat ggg cacaaa ttt tct gtc agt gga gag 96 Glu Leu Asp Gly Asp Val Asn Gly His LysPhe Ser Val Ser Gly Glu 20 25 30 ggt gaa ggt gat gca aca tac gga aaa cttacc ctt aaa ttt att tcc 144 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu ThrLeu Lys Phe Ile Ser 35 40 45 act act gga aaa cta cct gtt cca tgg cca acactt gtc act act ttc 192 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr LeuVal Thr Thr Phe 50 55 60 act tat ggt gtt caa tgc ttt tca aga tac cca gatcat atg aaa cgg 240 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp HisMet Lys Arg 65 70 75 80 cat gac ttt ttc aag agt gcc atg ccc gaa ggt tatgta cag gaa aga 288 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr ValGln Glu Arg 85 90 95 act ata ttt ttc aaa gat gac ggg aac tac aag aca cgtgct gaa gtc 336 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg AlaGlu Val 100 105 110 aag ttt gaa ggt gat acc ctt gtt aat aga atc gag ttaaaa ggt att 384 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu LysGly Ile 115 120 125 gat ttt aaa gaa gat gga aac att ctt gga cac aaa ttggaa tac aac 432 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu GluTyr Asn 130 135 140 tat aac tgc cac tgt gta tac atc atg gca gac aaa caaaag aat gga 480 Tyr Asn Cys His Cys Val Tyr Ile Met Ala Asp Lys Gln LysAsn Gly 145 150 155 160 atc aaa gtt aac ttc aaa att aga cac aac att gaagat gga agc gtt 528 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu AspGly Ser Val 165 170 175 caa cta gca gac cat tat caa caa aat act cca attggc gat ggc cct 576 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile GlyAsp Gly Pro 180 185 190 gtc ctt tta cca gac aac cat tac ctg tgc aca tgctct gcc ctt tcg 624 Val Leu Leu Pro Asp Asn His Tyr Leu Cys Thr Cys SerAla Leu Ser 195 200 205 aaa gat ccc aac gaa aag aga gac cac atg gtc cttctt gag ttt gta 672 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu LeuGlu Phe Val 210 215 220 aca gct gct ggg att aca cat ggc atg gat gaa ctatac aaa taa 717 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys225 230 235 11 238 PRT Artificial sequence Variant derived from A.victoria 11 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile LeuVal 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val SerGly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys PheIle Ser 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val ThrThr Phe 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His MetLys Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr ValGln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr ArgAla Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile GluLeu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly HisLys Leu Glu Tyr Asn 130 135 140 Tyr Asn Cys His Cys Val Tyr Ile Met AlaAsp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile ArgHis Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr GlnGln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp AsnHis Tyr Leu Cys Thr Cys Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn GluLys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala GlyIle Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 12 717 DNAArtificial sequence Variant derived from A. victoria 12 atg agt aaa ggagaa gaa ctt ttc act gga gtt gtc cca att ctt gtt 48 Met Ser Lys Gly GluGlu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 gaa tta gat ggtgat gtt aat ggg cac aaa ttt tct gtc agt gga gag 96 Glu Leu Asp Gly AspVal Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt gaa ggt gat gcaaca tac gga aaa ctt acc ctt aaa ttt att tcc 144 Gly Glu Gly Asp Ala ThrTyr Gly Lys Leu Thr Leu Lys Phe Ile Ser 35 40 45 act act gga aaa cta cctgtt cca tgg cca aca ctt gtc act act ttc 192 Thr Thr Gly Lys Leu Pro ValPro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 tct tat ggt gtt caa tgc ttttca aga tac cca gat cat atg aaa cgg 240 Ser Tyr Gly Val Gln Cys Phe SerArg Tyr Pro Asp His Met Lys Arg 65 70 75 80 cat gac ttt ttc aag agt gccatg ccc gaa ggt tat gta cag gaa aga 288 His Asp Phe Phe Lys Ser Ala MetPro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act ata ttt ttc aaa gat gac gggaac tac aag aca cgt gct gaa gtc 336 Thr Ile Phe Phe Lys Asp Asp Gly AsnTyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggt gat acc ctt gttaat aga atc gag tta aaa ggt att 384 Lys Phe Glu Gly Asp Thr Leu Val AsnArg Ile Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaa gat gga aac attctt gga cac aaa ttg gaa tac aac 432 Asp Phe Lys Glu Asp Gly Asn Ile LeuGly His Lys Leu Glu Tyr Asn 130 135 140 tat aac tgc cac tgt gta tac atcatg gca gac aaa caa aag aat gga 480 Tyr Asn Cys His Cys Val Tyr Ile MetAla Asp Lys Gln Lys Asn Gly 145 150 155 160 atc aaa gtt aac ttc aaa attaga cac aac att gaa gat gga agc gtt 528 Ile Lys Val Asn Phe Lys Ile ArgHis Asn Ile Glu Asp Gly Ser Val 165 170 175 caa cta gca gac cat tat caacaa aat act cca att ggc gat ggc cct 576 Gln Leu Ala Asp His Tyr Gln GlnAsn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt tta cca gac aac cattac ctg tgc aca tgc tct gcc ctt tcg 624 Val Leu Leu Pro Asp Asn His TyrLeu Cys Thr Cys Ser Ala Leu Ser 195 200 205 aaa gat ccc aac gaa aag agagac cac atg gtc ctt ctt gag ttt gta 672 Lys Asp Pro Asn Glu Lys Arg AspHis Met Val Leu Leu Glu Phe Val 210 215 220 aca gct gct ggg att aca catggc atg gat gaa cta tac aaa taa 717 Thr Ala Ala Gly Ile Thr His Gly MetAsp Glu Leu Tyr Lys 225 230 235 13 238 PRT Artificial sequence Variantderived from A. victoria 13 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly ValVal Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His LysPhe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys LeuThr Leu Lys Phe Ile Ser 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp ProThr Leu Val Thr Thr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg TyrPro Asp His Met Lys Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met ProGlu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly AsnTyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu ValAsn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly AsnIle Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Cys His Cys ValTyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val AsnPhe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu AlaAsp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val LeuLeu Pro Asp Asn His Tyr Leu Cys Thr Cys Ser Ala Leu Ser 195 200 205 LysAsp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235

We claim:
 1. A mutant Aequorea victoria green fluorescent protein (GFP),with a fluorescence spectrum that is sensitive to redox status, whereinat least one of the residues at position 147 or 149, and at least one ofthe residues at position 202 or 204, is mutated from the wild-type aminoacid to cysteine.
 2. The mutant GFP of claim 1, selected from the groupconsisting of GFPs in which: residue 147 and residue 202 are cysteine;residue 147 and residue 204 are cysteine; residue 149 and residue 202are cysteine; residue 149 and residue 204 are cysteine; and residues147, 149, 202 and 204 are each cysteine.
 3. The mutant GFP of claim 1,further comprising a mutation at position
 65. 4. The mutant GFP of claim3, wherein the mutation at position 65 is S65T.
 5. The mutant GFP ofclaim 1, wherein the fluorescence spectrum is also pH sensitive.
 6. Themutant GFP of claim 1, further comprising a mutation at position
 48. 7.The mutant GFP of claim 6, wherein the mutation at position 48 is C48S.8. The mutant GFP of claim 1, wherein said mutant protein comprises thefollowing mutations compared to wild-type GFP: C48S, S65T, N149C, andS202C.
 9. The mutant GFP of claim 1, wherein said mutant proteincomprises the following mutations compared to wild-type GFP: C48S, S65T,N149C, and Q204C.
 10. The mutant GFP of claim 1, wherein said mutantprotein comprises the following mutations compared to wild-type GFP:C48S, S147C, and Q204C.
 11. The mutant GFP of claim 1, wherein saidmutant protein comprises the following mutations compared to wild-typeGFP: C48S, S147C, and S202C.
 12. The mutant GFP of claim 1, wherein saidmutant protein comprises the following mutations compared to wild-typeGFP: C48S, S65T, S147C, N149C, S202C, and Q204C.
 13. The mutant GFP ofclaim 1, wherein said mutant protein comprises the following mutationscompared to wild-type GFP: C48S, S147C, N149C, S202C, and Q204C.
 14. Anisolated nucleic acid molecule encoding the mutant GFP of claim
 1. 15.An isolated nucleic acid molecule encoding the mutant GFP of any one ofclaims 2-13
 16. The isolated nucleic acid molecule of claim 14,comprising an expression control sequence.
 17. A recombinant nucleicacid comprising the nucleic acid molecule of claim 14 functionallylinked to a promoter.
 18. A host cell comprising a nucleic acidaccording to claim
 14. 19 The host cell of claim 18 wherein the hostcell is a bacterial cell, a plant cell, or an animal cell.
 20. The hostcell of claim 18 wherein said host cell is a mammalian cell.
 21. Amethod of analyzing an oxidation-reduction condition of or in a cellcomprising: expressing the mutant GFP of claim 1 in the cell; andmeasuring a fluorescence signal from the mutant GFP.
 23. The method ofclaim 21, wherein the mutant GFP is expressed as a fusion protein 24.The method of claim 21, further comprising analyzing a pH condition ofor in the cell using the mutant GFP.
 25. The mutant GFP of claim 1,comprising an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO; 9, SEQ ID NO: 11, or SEQ ID NO: 13.